2-amino-2-methylpropanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

• Animal fertility data on AMP-HCl (see Toxicity to reproduction /IUCLID § 7.8.1):

Two GLP and guideline-compliant studies are available, where rats were fed diets including AMP-HCl (neutralized AMP, 2-Amino-2-methyl-1-propanol hydrochloride, CAS N° 3207-12-3). Results are summarized below.

Table 1: Overview of animal fertility studies with AMP-HCl

Reference	Study design		Fertility/reproduction			Parental/general toxicity
Author year	Guideline, nb rats/sex at NOAEL	Nb treatment weeks (F0)	Adverse effects; LOAEL	NOAEL	Dietary AMP concentration at LOAEL; corresponding pH if not neutralized	Adverse effects; LOAEL	NOAEL	Dietary AMP concentration at LOAEL; corresponding pH if not neutralized
Millard 2021	OECD 443, 30 (F0) or 23-26 (F1)	M/F: 18	M/F: none; N/A	M/F: >= 142	M/F: N/A	M: hepatocellular vacuolation (minimal to moderate), lower incidence and severity in F1 than F0 generation ; 142 F: none; N/A	M: 71 F: 142	M: 0.22-0.34 % w/w; 10.9-11.1
Carney 2005	OECD 421, 12	M: 5 F: <8	M: none; N/A F: post-implantation loss (embryo resorption), litter loss; 213	M: >=710 F: 71	F: 0.22-0.26 % w/w; 10.9-11.0	M: hepatocellular vacuolation (up to readily observed); 710 F: hepatocellular vacuolation (up to readily observed); 71	M: 213 F: < 71	F: 0.09-0.12 % w/w; 10.6-10.7

Bold: key study; all NOAEL/LOAEL values in mg AMP/kg bw/day; M: males; F: females; N/A: not applicable; red: excess pH, MTD exceeded if AMP hadn't been neutralized

The OECD 421 study is much less robust than the OECD 443 study. Indeed, it includes 2-2.5 times less animals at NOAEL and treatment period is 2.2-3.6 times shorter. In addition, three OECD 414 studies are available with much more robust information on development (see below, Effects on development). Therefore, the OECD 421 study is supportive while the OECD 443 study is the key study.

The OECD 421 study showed that AMP-HCl has the potential to induce post-implantation loss in rats at 213 mg AMP/kg bw/day. This effect consists in early embryo resorption leading to partial or complete litter loss. In the OECD 443 study carried out in same strain of rats with the same test item and administration method, no post-implantation loss occurred at the top-dose of 142 mg AMP/kg bw/day, which is the overall NOAEL for this effect, proving the existence of a threshold.

All adverse effects observed in the OECD 443 and 421 studies occurred at dose-levels (LOAELs) which cannot be reached with AMP (base) as diet pH would have ranged 10.6-11.1 (see red values in table 1) compared to a tolerable maximum of 9 (Turner 2011, see IUCLID section 4.20). The LOAELs ranging 71 to 213 mg AMP/kg bw/day in both studies could only be reached by testing a different chemical (AMP hydrochloride, CAS N° 3207-12-3) with lower pH. This represents artificial, supra-guideline dose maximization as test item neutralization is not required by OECD guidelines.

Reproductive effects only occurred in the OECD 421 study. In this study, liver toxicity occurred at a 3-fold lower dose-level than reproductive effects. Mechanistic studies (summarized in "Mode of Action Analysis / Human Relevance Framework" below) investigating liver toxicity (hepatocyte vacuolation) and reproductive effects (post-implantation loss) suggested that these two effects share a common pathway: they are both associated with or correlated to, perturbation of choline uptake and choline metabolism into phospholipids.

Taken together, these facts and considerations allow to conclude that the reproductive effects observed after supra-guideline dosing with AMP-HCl (CAS N° 3207-12-3) and triggered by general toxicity in presence of liver toxicity, do not warrant a reproductive toxicity classification for AMP (CAS N° 124-68-5). This is further detailed below under "Justification for classification or non-classification‌".

 

• Animal necropsy of reproductive organs after ingestion of AMP-HCl /AMP /AMP neutralized (see Repeated dose toxicity /IUCLID § 7.5.1 and 7.8.3):

Multiple oral studies were carried out in dogs (4, 14, 26 and 52 weeks), rat (5 days, 2, 5-8, 8, 3x13 and 18 weeks), mouse (8 weeks), rabbits (3 weeks) and monkeys (5 days). None of them mentioned any post-mortem effects on reproductive organs in terms of organ weights or gross and microscopic findings. However, corresponding investigations were not present or complete in all these studies, and only the two rat studies tabulated above (OECD 443: 18 weeks; OECD 421: 5-8 weeks) covered the implantation key event.

 

• Human data on pamabrom (see Other studies /IUCLID § 7.8.3 and Exposure related observations in humans, IUCLID § 7.10.3):

Pamabrom is an Over The Counter (OTC) diuretic agent which is an AMP salt of 8-bromotheophylline, CAS No. 606-04-2.
It is an equimolar mixture of 74.4% w/w 8-Bromotheophylline (CAS No. 10381-75-6) and 25.6% w/w 2-amino-2-methylpropan-1-ol (AMP, CAS No. 124-68-5). Clinical data on this test material can be used for AMP safety assessment based on the AMP/Pamabrom bioequivalence study (see Basic Toxicokinetics /IUCLID §7.1.1) which indicated that Pamabrom's AMP, and AMP as is, were equivalent in terms of AUC0-t, AUC0-inf and AUC0-168h. Pamabrom doses can be converted into equivalent AMP doses based on the AMP content of 25.6% w/w in this drug and assuming a patient weight of 70 kg when not indicated.

A) Oral clinical studies:

Six clinical studies are available on pamabrom. NOAELs are expressed in AMP based on pamabrom posology and AMP content. In all cases the below-listed NOAELs were the maximum tested dose:

1) Trial in severe cardiac failure patients (Doherty et al, 1953): No clinical effects related to reproduction among n=19 patients. The NOAEL was >= 2.2 mg AMP/kg bw/day x 2 to 17 weeks depending on patient.

2) Trial in women with premenstrual tension - Preliminary study (McGavack et al, 1956): No adverse effect in n=9 women. The NOAEL was >= 2.9 mg AMP/kg bw/day x 4 weeks.

3) Trial in women with premenstrual tension - Main study (McGavack et al, 1956): No adverse effect in n=43 women. The NOAEL was >= 1.5 mg AMP/kg bw/day x 2 weeks (mean: variable
treatment regime).

4) Trial in women with primary dysmenorrhea (Ortiz et al, 2016): No clinical effects related to reproduction in n=189 patients. The NOAEL was >= 0.32 mg AMP/kg bw/day x 3 days.

5) Trial in pregnant women (Patterson, 1958): No adverse effects in n=38 pregnant women with edema classified as mild pre-eclampsia. The NOAEL was >= 5.9 mg AMP/kg bw/day x 1 week (second treatment cycle), and >= 2.9 mg AMP/kg bw/day x 2 weeks (both treatment cycles in patients treated twice).

6) Trial in pregnant women (James et al, 1957): No adverse effects in n=180 pregnant women with edema treated for unknown duration (Klimisch 4 study). The max. NOAEL was >= 2.2 mg AMP/kg bw/day, and in most patients it was >=1.6 mg AMP/kg bw/day.

Considering all clinical trials, the most robust human NOAEL for repeat-dose toxicity comes from study 2) with monitoring of hematology and blood biochemistry. The NOAEL was >= 2.9 mg AMP/kg bw/day x 4 weeks.

B) 70 years of safe clinical use - Pharmacovigilance argument:

In the US, pamabrom is used since early 1950's so it has a track-record of >70 years of safe use in an ill population. Based on the National Library of Medicines database, at least 20 US OTC drugs currently contain pamabrom (full list provided in study summary). Their claims include back/leg/joint pain and edema (1 drug which may thus be used during pregnancy) and premenstrual and menstrual pain/discomfort/edema (19 drugs which may thus be used in women of child-bearing age before pregnancy). The only mention related to reproduction is a generic statement used for almost all drugs: "If pregnant or breast-feeding, ask a health professional before use." Based on contents and posology for each drug, AMP has a human NOAEL of >=0.73 mg AMP/kg/day for up to 10-day treatment cycles, repeated over periods.

C) Overall human oral NOAEL for reproduction:

No adverse reproductive effect was ever reported during 70 years of clinical trials and medication with pamabrom based on 5 publications (6 trials) and posology of 20 OTC drugs. The most robust human oral NOAEL covering reproduction comes from study 5 in 38 pregnant women (Patterson, 1958): >= 5.9 mg AMP/kg bw/day x 1 week and >= 2.9 mg AMP/kg bw/day x 2 weeks. All other studies and OTC drug posology confirm AMP NOAEL values in the mg/kg bw/day range, always the highest tested dose whatever the treatment duration (3 days to 17 weeks + one trial where treatment duration was not indicated). To note, the 2-week reproductive NOAEL is the same value as the human 4-week repeat-dose toxicity NOAEL (see IUCLID § 7.5), although it does not come from the same publication, authors, year and target population. This shows that at realistic human exposure doses, AMP has neither repeat-dose nor reproductive toxic effects even when ingested daily.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Route:
The route of administration was oral (dietary) because this was a potential nature of exposure to humans. Historically, this route has been used extensively for studies of this nature.

Dose-levels:
In a previous OECD 421 study (Carney et al, 2005), at the high-dose level, all 12 pregnant females showed evidence of complete litter resorption (100% postimplantation loss), while at 300 mg/kg/day, postimplantation loss was 70% (vs. 10% in controls). Effects associated with, or secondary to the postimplantation loss increase at 300 mg/kg/day, included decreased litter size, increased pup body weight, and decreased gestation body weight and body weight gain. There were no treatment-related effects on reproductive performance in the 100 mg/kg/day group. The no-observed-effect level (NOEL) for general toxicity in males was 300 mg/kg/day, while the general toxicity NOEL for females could not be determined, based upon the presence of very slight microscopic liver effects. The NOEL for reproductive effects was considered to be 100 mg/kg/day.

Specific details on test material used for the study:

99.2% pure = high-purity grade

Based on molecular weights of this equimolar salt, AMP-HCl (CAS 3207-12-3, MW = 125.60) contains 71.0% AMP (CAS 124-68-5, MW = 89.14). Therefore AMP-HCl doses can be converted into AMP doses using a correction factor of x0.71.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has
reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be suscept
ible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation (P) 11 wks
- Weight at study initiation: (P) Males: 346-456 g; Females: 209-294 g
- Fasting period study initiation: No
- Housing: On arrival, the F0 animals were group housed (2 to 3 animals of the same sex). During
cohabitation, the F0 animals were paired for mating in the home cage of the male. Following the
breeding period, animals were individually housed. Following weaning (F1), animals were group hous
ed (2 to 3 animals of the same sex) until euthanasia.
Animals were housed in solid-bottom cages containing appropriate bedding equipped with an autom
atic watering valve. Animals were separated during designated procedures/activities. Each cage
was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s),
dosage level, and sex. Cages were arranged on the racks in group order.
Whenever possible, animals were socially housed for psychological/environmental enrichment and
were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral
health.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal). Ad libitum, exception:
prior to blood sampling for clinical biochemistry, animals were fasted overnight
- Water: ad libitum; municipal tap water after treatment by reverse osmosis and ultraviolet irradiation.
- Acclimation period:13 days
ENVIRONMENTAL CONDITIONS (SET TO MAINTAIN):
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark /hrs light):12/12
The feed was analyzed by the supplier for nutritional components and environmental contaminants.
Periodic analysis of the water was performed. Results of the analysis are on file at the Test Facility.
It is considered that there were no known contaminants in the feed and the water that would interfere
with the objectives of the study.
IN-LIFE DATES:
From: 22 Oct 2019 To: 12 May 2020

Route of administration:

oral: feed

Vehicle:

other: other: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal)

Details on exposure:

DIET PREPARATION
For administration to Group 1 control animals, an appropriate amount of PMI Nutrition International,
LLC Certified Rodent LabDiet® 5002 was weighed out weekly and placed in a labeled bag.
For administration to Group 2, 3 and 4 animals, an appropriate amount of the test substance for each
group was added to PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 on a weight/
weight basis, and mixed to form a premix. The remainder of rodent feed to achieve the desired co
ncentration added to the pre-mix, after which the diet was blended to achieve a total batch of homog
eneous diet at the appropriate concentration/group. The test diets were prepared approximately weekl
y, or as needed, and stored at room temperature.
ADAPTATIONS DURING GESTATION AND LACTATION
During gestation and lactation, concentration of the test substance in the diet were adjusted based on
historical control data for body weights and food consumption data for gestating and lactating females
in order to compensate for the higher caloric demand on maternal animals during these periods.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
Analyses were performed by an ultra-high performance liquid chromatography method using a validated analytical procedure. Analysis to demonstrate the stability and homogeneity of test diet admixes between 50 and 15,000 ppm for at least 4 and 10 days under room temperature and refrigerated (target of 5°C) conditions has been previously established by Charles River Ashland.
Homogeneity and stability of the test substance in dietary preparations prepared as low as 50 ppm were established prior to administration to study animals by Charles River Ashland.
SAMPLING
Dose formulation samples were taken weekly to monthly for the analysis of concentration (all groups). Samples for homogeneity analysis (groups 2-4) were taken from the first prepared batch only. Due to the adjustment of concentrations of the test substance in the diet beginning on PND 21, homogeneity and stability of the test substance in dietary preparations prepared as low as 50 ppm were established prior to administration of this concentration to study animals by Charles River Ashland.
ACCEPTANCE CRITERIA
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was within 20% or less at a concentration that is within the acceptable limits (80% to 120% of the target concentrations). After acceptance of the analytical results, backup samples were discarded.

Duration of treatment / exposure:

F0 FEMALES: F0 females were administered the test substance continuously in the diet for a minimum of 70 consecutive days (10 weeks) prior to mating, during mating (max 2 weeks), gestation and lactation (~3 weeks each) and until scheduled necropsy on study week 18. Total treatment duration: 18 weeks.

F0 MALES: F0 males were administered the test substance continuously in the diet over the same period as females, until scheduled necropsy on study week 18. Total treatment duration: 18 weeks.

COHORT 1A and 1B: The offspring selected for the F1 generation was indirectly exposed via F0 females, in utero and then potentially via lactation (~3 weeks each). Then they were administered test substance in the diet from weaning until fasting for scheduled necropsy on PND 91 [Cohort 1A] or 98 [Cohort 1B]). Total treatment duration: 6 weeks indirect exposure followed by 13-14 weeks direct exposure.

Frequency of treatment:

continuously through diet

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

achieved dose-levels in F0 (mg/kg/d):
50-51 in males until sacrifice and in females until end of mating
42 in females during gestation
45 in females during lactation
51-52 in M/F during F1 generation

containing 35 mg/kg bw/day AMP

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

achieved dose-levels in F0 (mg/kg/d):
100-103 in males until sacrifice and in females until end of mating
80 in females during gestation
91 in females during lactation
103 in M/F during F1 generation

containing 71 mg/kg bw/day AMP

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

achieved dose-levels in F0 (mg/kg/d):
197-203 in males until sacrifice and in females until end of mating
164 in females during gestation
181 in females during lactation
204 in M/F during F1 generation

containing 142 mg/kg bw/day AMP

No. of animals per sex per dose:

25 in F0 groups 1, 2, 3
30 in F0 group 4 (200 mg/kg/day)
21-22 (M & F) in F1 cohort 1A groups 1, 2, 3
26 M + 24 F in F1 cohort 1A group 4 (200 mg/kg/day)
19-22 (M & F) in F1 cohort 1B groups 1, 2, 3
25 M + 23 F in F1 cohort 1B group 4 (200 mg/kg/day)

Control animals:

yes, plain diet

Details on study design:

-- Rationale for animal assignment (if not random):
F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at extremes of body weight range were not assigned to groups. To reduce variability among the F1 litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. For the F1 generation, 2 F1 pups/sex/litter from all available litters (up to 25 or 30 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts. Cohorts 1A and 1B were assigned to reproductive/developmental toxicity testing. Animals assigned to Cohort 1B were maintained on study for possible breeding when the animals were between 90 and 120 days of age to generate an F2 generation; however, additional breeding was not required on this study. In addition, if there were an insufficient number of pups to fill a designated cohort, Cohort 1A was given priority over Cohort 1B.

Offspring allocation by cohort:
Cohort 1A: 1 pup/sex/litter/group for evaluation of primary reproductive/developmental toxicity assessment.
Cohort 1B: 1 pup/sex/litter/group for evaluation of follow-up reproductive assessment (not required)

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study
- General health/mortality and moribundity
DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once daily throughout the study
BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout the study and prior to necropsy Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 14, and 21. Body weights were collected prior to and after fasting.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly throughout the study, except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 14, and 21. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The mean amounts of test substance consumed (mg/kg bw/day) by each sex per dose group were calculated from the mean food consumed (g/kg bw/day) and the appropriate target concentration of test substance in the food (mg/kg).
THYROID HORMONE ANALYSIS
Animals were fasted overnight prior to blood collection. Blood samples for thyroid hormone analyses (Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH)) were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from a jugular vein into tubes without anticoagulants. Samples were collected from 10 animals/sex/group in week 18.
CLINICAL PATHOLOGY
Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and serum chemistry were collected from a jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants. Samples were collected from 10 animals/sex/group in week 18. Hematology, coagulation, serum chemistry and urinalysis parameters were in line with OECD TG 443 guidance.

Oestrous cyclicity (parental animals):

Vaginal lavages were performed for cytological evaluation to determine the stage of the estrous cycl
e of each F0 female on the day of necropsy, and daily for 14 days prior to cohabitation and continui
ng until evidence of mating or until the end of the mating period. The average cycle length was calc
ulated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [
D] from estrus [E] or proestrus [P], beginning 14 days prior to initiation of the mating period and
continuing until the detection of evidence of mating). Estrous cycle length was determined by counti
ng the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The
cycle during which evidence of mating was observed for a given animal was not included in the mean
individual estrous cycle length calculation. Definitions based on findings for each female:
Regular cycling (RC): animal has at least 6 days of data collected and displays 1 complete cycle with
no cycles >5 days in duration, no cycles with ≥3 consecutive days of P and/or E, and no cycles with
<4 days in duration.
Irregular cycling (IC): animal does not display 1 complete cycle but has ≥1 E and/or P and a partial c
ycle >5 days, the animal has ≥E present and either ≥1 cycle (complete or partial) >5 days in duration or ≥1 cycle with ≥3 consecutive days of P and/or E, ≥1 cycle <4 days in duration, or 1 irregular cycle
and 1 regular cycle.
Non-cycling (NC): no E or P present on any days of estrous cycle determination and at least 5 days
of data collected.
Insufficient data (ID): animal does not display ≥1 complete cycle but has ≥1 E and/or P and a partial
cycle of ≤5 das, no E present on any day of cycle determination and ≤4 days of data collected, or ≥1
E or P present and only 1–4 days of data collected.
% Cycling = (RC + IC+ ID)/(RC + IC+ ID + NC)
% Cycling regularly = RC/(RC+IC)

Sperm parameters (parental animals):

For all surviving F0 animals, the following assessments were performed:
-Sperm motility was determined as soon as possible after euthanasia. After weighing the right cauda
epididymis, a sample of sperm was taken for determination of sperm motility. Analysis of a minimum
of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed .
The motility score (percent) for motile (showing motion only) and progressively motile (showing net fo
rward motion) sperm was reported:
% Motile (or progressively motile) sperm = No. of Motile (or Progressively Motile) Sperm/Total No. of
Sperm counted x100
-Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation
technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic,
etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
-After weighing, the left testis and cauda epididymis from all males were stored frozen, homogenized,
and analyzed for determination of homogenization resistant spermatid count and calculation of sperm
production rate.
Sperm Production Rate = No. of Sperm per Gram Tissue/6.1 Days
with 6.1 days the rate of turnover of the germinal epithelium

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and
discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring until weaning (PND21):
- General health/mortality and moribundity twice daily. A daily record of litter size was maintained. The
following parameters were recorded: Mean live litter size; Postnatal survival between birth and PND0
or PND4 (Pre-selection; % per litter); Postnatal survival for all other intervals (% per litter); Total litter
loss determined when the last pup in the litter was found dead or euthanized in extremis prior to the
scheduled euthanasia.
- Clinical observations: on PND 1, 4, 7, 14, and 21. Any abnormalities in nesting and nursing behavior
were recorded.
- Sex determination on PND 0, 4, 14, and 21
- Body weights: pups were weighed individually on PND 1, 4, 7, 14, and 21
- Anogenital distance: for all pups measured on PND 1. Anogenital distance was defined as the
distance from the caudal margin of the anus to the caudal margin of the genital tubercle.
-Thoracic nipples/areola: all male pups on PND 13
-Thyroid hormone analysis in culled/nonselected pups (PND4/PND21): blood samples for thyroid
hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in
thyroid hormone levels) via cardiac puncture of animals anesthetized by inhalation of isoflurane (PND
4 culled pups) or via cardiac puncture (PND 21 non-selected pups - 10/sex/group). Samples collected
from culled pups were pooled by litter until a total of 10 samples/dosage level were obtained. To the
extent possible, samples from the first 10 litters at each dosage level with sufficient numbers of culled
pups were used.
From weaning (PND21) onwards (Cohorts 1A and 1B)
CAGE SIDE OBSERVATIONS:
- General health/mortality and moribundity twice daily.
CLINICAL OBSERVATIONS
- Detailed clinical observations once daily
BODY WEIGHT
- Individual body weight weekly following weaning.
- each female on the day of acquisition of vaginal patency
- each male on the day of acquisition of balanopreputial separation
- a fasted weight was recorded on the day of necropsy for Cohort 1A animals (PND91)
FOOD CONSUMPTION, FOOD EFFICIENCY AND COMPOUND INTAKE
-Food consumption quantitatively measured weekly.
-Food efficiency (body weight gained as a percentage of food consumed) was calculated
-Mean amounts of test substance consumed (mg/kg bw/day) by each sex per dose group calculated
from the mean food consumed (g/kg bw/day) and the appropriate target concentration of test substa
nce in the food (mg/kg).
BALANOPREPUTIAL SEPARATION
-Each male was observed for balanopreputial separation beginning on PND 35. Examination of the
males was continued daily until balanopreputial separation was present, and the age of attainment
was recorded. Body weights were recorded at the age of attainment of this landmark. In addition,
the appearance of a partial and complete balanopreputial separation or a persistent thread of tissue
between the glans and prepuce was recorded.
VAGINAL PATENCY
Each female was observed for vaginal perforation beginning on PND 25. Examination of the females
was continued daily until vaginal patency was present, and the age of attainment was recorded. Bo
dy weights were recorded at the age of attainment of this landmark. In addition, the appearance of a
small “pin hole”, a vaginal thread, and complete vaginal opening were recorded.
ESTRUS CYCLE DETERMINATION
- Beginning on the day vaginal opening was observed, vaginal lavages were performed daily and
the slides were evaluated microscopically to determine the stage of the estrous cycle of each female
until the first sign of estrus (cornified cells) was observed. The age of first vaginal estrus after vaginal
opening was recorded.
-Oestrus cyclicity: Vaginal lavages were performed daily for all F1 females assigned to Cohort 1A and
the slides were evaluated microscopically to determine the stage of the estrous cycle of each female
for 2 weeks during PND 75–91 (the day of necropsy). The average cycle length was calculated and
reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D]
from estrus [E] or proestrus [P]). Estrous cycle length was determined by counting the number of da
ys from the first M or D in a cycle to the first M or D in a subsequent cycle. At the end of the study, the
overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling,
or insufficient data.
% Cycling = (RC + IC+ ID)/(RC + IC+ ID + NC)
with ID: animals categorized as 'Insufficient Data' were included in calculation only if an E (Estrus) or
P (Proestrus) was present on any of the evaluation days. Animals categorized as 'Insufficient Data
that did not have an E or P present were not included in the calculation.
% Cycling regularly = RC/(RC+IC)
THYROID HORMONE ANALYSIS (Cohort 1A)
At PND91, animals were fasted overnight and blood samples for thyroid hormone analyses (Thyroxine
(Total T4) and Thyroid Stimulating Hormone (TSH)) were collected (prior to 1200 hours in order to
avoid normal diurnal fluctuation in thyroid hormone levels) from a jugular vein into tubes without anti
coagulants. Samples were collected from 10 animals/sex/group
CLINICAL PATHOLOGY (Cohort 1A)
At PND91, animals were fasted overnight prior to blood collection. Urine was collected overnight using
metabolism cages. Blood samples for hematology and serum chemistry were collected from the jug
ular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the in
ferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.
K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was
used for samples collected for clotting determinations. Samples for serum chemistry were collected
without anticoagulants.
Blood and urine samples were analyzed for the clinical pathology parameters specified in the tables.

Postmortem examinations (parental animals):

SACRIFICE
All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation following the selection of the F1 generation in week 18
GROSS NECROPSY
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of implantation sites and former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
HISTOPATHOLOGY / ORGAN WEIGHTS
ORGAN WEIGHT: Organs of F0 animals were collected in line with OECD TG 443. Organ weights were not recorded for animals euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
HISTOPATHOLOGY
Tissues collected from all animals in the control and high-dose groups and from all animals euthanized in extremis, as well as gross lesions from all animals in all groups, and reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were preserved, processed and evaluated according to OECD TG 443. Processing of the testes, epidididymes, and ovaries were performed as noted below.
Testis and epididymis: Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Ovary: Five (5) sections were taken approximately 100 μm apart from the inner third of each ovary of F0 females suspected of reduced fertility. In addition, a single section was taken from remaining F0 females for a qualitative bilateral evaluation of each ovary. For females euthanized in extremis, a single section from each ovary was qualitatively evaluated. For any F0 female suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles. Uterine and ovarian histopathology were considered in light of the terminal estrous stage.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation
included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Postmortem examinations (offspring):

SACRIFICE, ORGAN WEIGHT AND HISTOPATHOLOGY
F1 UNSCHEDULED DEATHS
No F1 offspring died during the course of the study.
F1 CULLED PUPS
On PND 4, culled pups were euthanized by exsanguination (those pups used for blood/thyroid co
llection) or an intraperitoneal injection of sodium pentobarbital. All remaining culled pups were di
scarded without examination.
Representative samples of trachea (with thyroid glands and all gross lesions were collected from 1 cu
lled pup/sex/litter and preserved.
F1 NONSELECTED as PARENTAL ANIMALS
The F1 offspring not selected as parental animals were sacrificed at on PND21 by exsanguination. Th
ese animals were subjected to a complete necropsy examination, with emphasis on developmental
morphology and organs of the reproductive system:
- Organ weights from 1 nonselected F1 pup/sex/litter: brain, spleen, thymus and thyroid (after
fixation). Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios
were calculated
-Representative samples of the following tissues collected from at least 1 nonselected F1 pup/sex/li
tter and preserved: brain, liver, ovaries, skin with mammary gland for females (for males correspond
ing section of skin was taken from the same anatomical area), spleen, testes (for females correspond
ing section of skin was taken from the same anatomical area), thymus, thyroid and all gross lesions.
COHORT 1A AND 1B
All surviving animals were euthanized by carbon dioxide inhalation.
Cohort 1A: PND 91
Cohort 1B: PHD 98
All animals were subjected to a complete necropsy examination, which included examination of the
external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and
the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the or
gans of the reproductive system.
The organs indicated in the OECD TG 443 were weighed at necropsy for all scheduled euthanasia an
imals. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (usin
g the terminal body weight) and organ to brain weight ratios were calculated.
Representative samples of the tissues identified in Tissue Collection and Preservation - F1 Cohort 1A
and 1B were collected from all animals and preserved in 10% neutral buffered formalin, unless otherw
ise indicated.
HISTOLOGY (COHORT 1A)
Tissues identified in OECD TG 443 from Cohort 1A animals in the control and high-dose groups as
well as gross lesions from all animals in all groups were embedded in paraffin, sectioned, mounted
on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and
ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with
PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. The f
ollowing regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas defe
rens was examined when possible.
Five (5) sections were taken approximately 100 μm apart from the inner third of each ovary from all F1
Cohort 1A females at the scheduled termination. In addition, a single section was taken from all F1
Cohort 1A females for a qualitative bilateral evaluation of each ovary.
The coagulating glands, ovaries, pituitary gland, prostate gland, seminal vesicles, testes with epid
idymides, uterus with cervix and vagina, and gross lesions from Cohort 1B animals were processed
to the block stage (in paraffin).
HISTOPATHOLOGY COHORT 1A
Tissues retained for microscopic examination (OECD TG 443) were evaluated from all animals in the
control and high dose groups and gross lesions were examined from all animals in all groups.
Histopathological examination of the testis included a qualitative assessment of the stages of spermat
ogenesis. For males, microscopic evaluation included a qualitative assessment of the relationships
between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of
the seminiferous tubules. The progression of these cellular associations defines the cycle of sperm
atogenesis. In addition, sections of both testes were examined for the presence of degenerative chan
ges (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, i
nflammatory changes, mineralization, and fibrosis). When possible, sections of the rete testis were
examined in the F1 Cohort 1A males.
For all F1 Cohort 1A females in the control and high-dose groups at scheduled termination, a q
uantitative histopathologic evaluation of multiple sections of the ovaries was conducted. This exami
nation included enumeration of the total number of primordial follicles primordial follicles. Uterine and
ovarian histopathology were considered in light of the terminal estrous stage.
SPERM ANALYSIS COHORT 1A
Immediately upon euthanasia, the right cauda epididymis was excised and weighed. A sample of
sperm was taken for determination of sperm motility. Analysis of a minimum of 200 motile and no
nmotile spermatozoa per animal (if possible) in all groups was performed. The motility score (percen
t) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was
reported:
% Motile (or progressively motile) sperm = No of Motile (or Progressively Motile) Sperm/Total No of
Sperm counted x100
Sperm morphology was evaluated by light microscopy of a sample of the right epididymis. Abnormal
forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential
count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and
analyzed for determination of homogenization resistant spermatid count and calculation of sperm pro
duction rate.
Sperm Production Rate = No. of Sperm per Gram Tissue/6.1 Days
with 6.1 days the rate of turnover of the germinal epithelium
Statistics
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were co
nducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% and
5%, comparing each test substance exposed group to the control group by sex.
Male and female mating, fertility, copulation, and conception indices were analyzed using the Chi sq
uare test with Yates’ correction factor.
Mean F0 and F1 adult (weekly, gestation, and/or lactation) and offspring body weights and body wei
ght changes, food consumption, food efficiency data, estrous cycle lengths, pre coital intervals, ge
station lengths, former implantation sites, live litter sizes, unaccounted for sites, numbers of pups
born, balanopreputial separation data (day of attainment and body weight), vaginal patency data (d
ay of attainment and body weight), time from vaginal patency to first estrus, anogenital distance (a
bsolute and relative to the cube root of body weight), numbers of nipples/areolae, absolute and rela
tive organ weights, sperm production rates, epididymal and testicular sperm numbers, ovarian primord
ial follicle counts, and clinical pathology and thyroid hormone data were subjected to a parametric
one way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05)
intergroup variance, Dunnett's test was used to compare the test substance exposed groups to the
control group.
Mean litter proportions (percent per litter) of postnatal pup survival and pup sexes at birth (percentage
of males per litter), percentages of motile and progressively motile sperm, and percentages of sperm
with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determi
ne intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s
test was used to compare the test substance exposed groups to the control group.

Reproductive indices:

Male mating index (%): Number of males with evidence of mating/Total No. of males used for mating x 100
Female mating index (%): Number of females with evidence of mating (or confirmed pregnancy)/Total
No. of females used for mating x 100
Male copulation index (%): No. of Males siring a Litter/No. of males with evidence of mating (or fema
les confirmed pregnancy) x100
Male fertility index (%): Number of males siring a litter/Total No. of males used for mating x 100
Female fertility index (%): Number of females with confirmed pregnancy/Total No. of females used for
mating x 100
Female conception index (%): No. of females with confirmed pregnancy/No. of females with evidence
of matiing (or confirmed pregnancy) x100
Gestation index (%): Number of females with living pups on Day 1/Number of pregnant females x 100
Individual gestation length was calculated using the date delivery was first observed.

Offspring viability indices:

Postnatal Survival Between Birth and PND0 or PND4 (% per Litter) = Sum of (Viable Pups/Litter
PND0 or PND4 [Pre-Selection]/No. of Pups Born/Litter) * x100/No. of Litters/Group
Postnatal Survival for all Other Intervals (% per Litter) = Sum of (Viable Pups/Litter at end of Interval
N/Viable Pups/Litter at Start of Interval N) * x100/No. of Litters/Group
where N = PND0-1, 1-4 (Pre-selection), 4 (Post-selection)-7, 7-14, 14-21 or 4 (Post-selection)-21
and * = Pups that were euthanized due to death of the dam were excluded from pup viability calcu
lations.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 50 mg/kg bw/day group was euthanized in extremis on Lactation Day 8 due to poor clinical condition, including clinical observations of hunched posture, a thin and pale body, red material around the nose, and pale and cool extremities noted up to 2 days prior to euthanasia. Uterine adhesions, and one mummified fetus and 2 severely autolyzed fetuses in utero were observed grossly. Histologically, there was evidence of a uterine infection (severe inflammation with intralesional bacteria) and secondary sepsis (inflammation on the surface of multiple abdominal and thoracic organs with bacteria, decreased lymphoid cellularity in lymphoid organs, increased macrophages in the spleen, and increased myeloid cellularity in the bone marrow). Therefore, the cause of death was determined to be sepsis, secondary to the retained fetuses. All other F0 animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects were noted at all determinations, except for the below.

Lower (statistically significant) mean body weight gains were noted in the 50, 100, and 200 mg/kg bw/day groups when the Lactation Days 1-21 cumulative interval was evaluated compared to the control group, albeit not in a clear dose-related manner. However, mean absolute body weights in these groups were unaffected throughout lactation; therefore, these differences were not considered test substance related.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects were noted at all determinations, except for the below.

Lower (statistically significantly) mean food efficiency was noted in the 50, 100, and 200 mg/kg bw/day groups during the Lactation Days 1-21 cumulative interval compared to the control group, albeit not in a clear dose-related manner (see attached table). However, mean absolute body weights in these groups were unaffected throughout lactation; therefore, these differences were not considered test substance related.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance exposure was associated with hepatocellular vacuolation in the liver of F0 generation males at ≥ 50 mg/kg bw/day with a dose-related increase in incidence and severity. This lesion was characterized by minimal to moderate vacuolation of hepatocellular cytoplasm by clear round variably sized vacuoles. The distribution was multifocal and centrilobular to random. Hepatocellular vacuolation was considered adverse at 200 mg/kg bw/day due to the moderate severity.

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related effects on any F0 reproductive performance endpoints (all indices, pre-coital interval, duration of gestation).

Three, 2, and 3 mating pairs in the control, 50, and 100 mg/kg bw/day groups, respectively, did not produce a litter. In the F0 generation, reproductive tissues from eight pairs of males and females with suspected reduced fertility were evaluated microscopically. The suspected reduced fertility was not associated with test substance-administration. Three animal pairs were in Group 1 (0 mg/kg bw/day), two pairs in Group 2 (50 mg/kg bw/day), and three pairs in Group 3 (100 mg/kg bw/day). One female each in Group 1, 2 and 3 had histologic findings in the ovary consistent with early reproductive senescence (increased numbers of atretic follicles, decreased numbers of corpora lutea), which can be occasionally seen in this age of rat (Vidal, 2017). The cause of the suspected reduced fertility in the other animals was not determined by light microscopic evaluation but was not considered related to the test substance in the absence of a dose-related response.

The mean gestation lengths in the 50, 100, and 200 mg/kg bw/day groups were 22.2, 21.8, and 22.1 days, respectively, compared to mean gestation lengths of 21.8 days in the concurrent control group; the differences were statistically significant in the 50 and 200 mg/kg/day groups. The higher mean gestation lengths in the 50 and 200 mg/kg/day groups were not considered test substance-related because changes were not dose-responsive, and/or the values were within the Charles River Ashland historical control data range (20.9 to 22.1 days). No signs of dystocia were noted at any exposure level.

In the 200 mg/kg/day group, the mean number of pups born (11.5 pups/dam) and live litter size
on PND 0 (11.3 pups/dam) were lower (not statistically significant) than controls (12.8 pups/dam for both) and the respective minimum mean values in the Charles River Ashland historical control data (12.1 and 11.9 pups/dam). No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

general/systemic toxicity

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

general/systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 71 mg/kg bw/day AMP

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

general/systemic toxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Before weaning: F1: Nine (4), 10(7), 9(6), and 18(10) pups (litters) in the control, 50, 100, and 200 mg/kg/day groups, respectively, were found dead or euthanized in extremis from PND 0 through the selection of the F1 generation. No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead.

Post-weaning: No effects during F1 generation.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Prior to weaning:
UNSCHEDULED DEATHS: No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead.

Cohort 1A:
There were 4 males in the 200 mg/kg/day group, 1 male in the 100 mg/kg/day group, 3 females in the 200 mg/kg/day group, 1 female in the 100 mg/kg/day group, and 1 control group female with small thyroid glands. This change was unilateral and did not have histologic correlates. Therefore, the small thyroid glands were considered unrelated to test substance administration.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A:
Microscopic findings noted in the F0 generation animals were observed at the end of PND 91 and are summarized in the attached table. The test substance was associated with a dose related increase in incidence and severity of hepatocellular vacuolation in the liver of F1 generation Cohort 1A males at ≥ 50 mg/kg bw/day. Histologically, the lesion was similar to, but at a lower incidence, than that described in the F0 generation males and was considered adverse due to the moderate severity seen in 1 male in the 200 mg/kg bw/day group.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significantly higher mean estrous cycle length was noted in the 200 mg/kg/day group compared to the concurrent control group. This difference was not considered test substance-related because the mean estrous cycle length in this group (4.8 days) was within the Charles River Ashland historical control data range (3.9 to 5.2 days) and the concurrent control group value (3.8 days) was below the minimum mean value in the historical control data.

no effect on serum concentrations of T4 and TSH for F1 males and females on PND 21 and in Cohort 1A.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction/development

Generation:

F1

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

general/systemic toxicity

Generation:

F1

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

general/systemic toxicity

Generation:

F1 (cohort 1A)

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 71 mg/kg bw/day AMP

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

general/systemic toxicity

Generation:

F1 (cohort 1A)

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Key result

Reproductive effects observed:

no

RESULTS ANALYTICAL VERIFICATION OF DOSES: The analyzed dietary formulations contained 83.2% to 115% of the test substance which was within the protocol-specified range of target concentrations for suspensions (80% to 120%) and were homogeneous. The test substance was not detected in the analyzed control group diet that was administered to the control group.

Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117 (2011), a second generation was not assessed on this study.

F0 / Incidence of treatment-related Liver Effects

Sex	Male				Female
Dose (mg/kg/day)	0	50	100	200	0	50	100	200
Liver (# examined)	25	25	25	30	25	0	0	30
Vacuolization, hepatocellular, multifocal, centrilobular to random:	0	4	11	24	0	-	-	0
minimal	0	4	10	10	0	-	-	0
mild	0	0	1	7	0	-	-	0
moderate	0	0	0	7	0	-	-	0

Bold type indicates the effects judged to be adverse.

 

F1A / Incidence of treatment-related Liver Effects

Sex	Male				Female
Dose (mg/kg/day)	0	50	100	200	0	50	100	200
Liver (# examined)	20	22	22	20	20	0	0	20
Vacuolization, hepatocellular, multifocal, centrilobular to random:	0	1	7	8	1	-	-	0
minimal	0	1	7	4	1	-	-	0
mild	0	0	0	3	0	-	-	0
moderate	0	0	0	1	0	-	-	0

Bold type indicates the effects judged to be adverse.

Conclusions:

In a rat dietary fertility toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP had no effect on fertility and development up to 142 mg AMP/kg bw/day. Parental NOAELs were 71 and 142 mg AMP/kg bw/day in males and females, resp.

Executive summary:

In an OECD 443 study, groups of 19 to 30 CD rats/sex/dose were fed diets supplying 0 (control), 50, 100, or 200 mg AMP-HCl/kg bw/day (containing 35, 71 and 142 mg AMP/kg bw/day, resp.) over two generations (F0 and F1 with cohorts 1A and 1B). Toxicologically relevant effects were limited to liver toxicity in male rats, with lower incidence and severity in F1A generation than in F0 generation: 

• At 142 mg AMP/kg bw/day, liver weights were 12% increased in F0 only, and minimal to moderate (adverse) hepatocellular vacuolation was seen in 24/30 F0 rats and 8/20 F1A rats;


• At 71 mg AMP/kg bw/day, non-adverse hepatocellular vacuolation was seen in 11/25 F0 rats (minimal to mild) and 7/22 F1A rats (minimal);


• At 35 mg AMP/kg bw/day, non-adverse minimal hepatocellular vacuolation was seen in 4/25 F0 rats and 1/22 F1A rats.

Based on liver changes, the NOAEL for parental/general toxicity was 71 and 142 mg AMP/kg bw/day in males and females, resp., and the male LOAEL was 142 mg AMP/kg bw/day. In absence of effect on reproduction and neonatal development, NOAELs for reproduction and neonatal development were 142 mg AMP/kg bw/day.

Below conclusions were drawn post-report by the registrant:

1) At the LOAEL (male F0: 142 mg AMP/kg bw/day), diet contained 3155-4801 ppm AMP-HCl (see report p. 54), i.e. 0.224-0.341 % w/w AMP. AMP was tested neutralized as AMP-HCl, CAS No. 3207-12-3. As is, AMP is alkaline (high-purity-grade: pKa = 9.70). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3189 ln(Concentration in % w/w) + 11.401], pH of high-purity-grade AMP solutions at 0.224-0.341 % AMP w/w would range 10.9-11.1. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is instead of AMP-HCl, the study's LOAEL could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines referenced by REACH do NOT require any neutralization or pH adjustment of test items, this study represents artificial dose maximization in excess of REACH principles, removing AMP's critical toxic effect which is pH-dependent non-specific toxicity.

2) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

see post-report notes in executive summary

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

99.7% pure = high-purity grade

Based on molecular weights of this equimolar salt, AMP-HCl (CAS 3207-12-3, MW = 125.60) contains 71.0% AMP (CAS 124-68-5, MW = 89.14). Therefore AMP-HCl doses can be converted into AMP doses using a correction factor of x0.71.

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

CD (Crl:CD(SD)IGSBR) rats were obtained from a commercial supplier and were approximately eight weeks of age at the time of study initiation. Each animal was evaluated by a laboratory veterinarian or a trained animal/toxicology technician, under the direct supervision of a lab veterinarian to determine their general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed 2-3 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately two weeks prior to the start of the study. They were offered a commercial diet and water ad libitum. During the study, animals were housed one per cage (prebreeding) or two per cage (one male and one female during breeding) in stainless steel cage in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Dams were housed one per cage (with their litter) in plastic cages provided with corn cob nesting material from approximately day 19 of gestation and throughout the lactation phase of the study. Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers.

Route of administration:

oral: feed

Vehicle:

other: feed

Details on exposure:

Groups of 12 male and 12 female CD rats were fed diets supplying 0 (control), 100, 300, or 1000 mg/kg/day of AMP HCl. Diets were prepared weekly.

Details on mating procedure:

Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered day 0 of gestation. The sperm or plug-positive (presumed pregnant) females then were separated from the male and returned to their home cages. If mating did not occur after two weeks, the animals were separated without further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples from the test diets were evaluated concurrently with the concentration verification analyses (see below) to ensure homogeneous distribution of the test material at the lowest and highest concentrations in the feed at least once during the study. Preliminary stability analyses of the test material in rodent diets at concentrations of 0.0005% and 0.005% and 5.0% were initiated prior to the start of the range-finding study. Analysis of all test diets from the first mix of the main study were initiated prior to the start of dosing using gas chromatography-mass spectrometry (GC-MS) incorporating an internal standard to determine target concentrations.

Duration of treatment / exposure:

Males were exposed for at least two weeks prior to breeding and continuing throughout breeding for 37 days. The females were exposed for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days).
Resulting duration of treatment: F0 males: 5 weeks, F0 females: <8 weeks, F1: <4 weeks (indirect via gestation and 4-day lactation)

Frequency of treatment:

continuous via diet

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

containing 71 mg/kg bw/day AMP

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

containing 213 mg/kg bw/day AMP

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

containing 710 mg/kg bw/day AMP

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

Groups of 12 male and 12 female CD rats were fed diets supplying 0 (control), 100, 300, or 1000 mg/kg/day of AMP HCl (0, 71, 213, 710 mg/kg/day AMP). Males were exposed for at least two weeks prior to breeding and continuing throughout breeding for 37 days. The females were exposed for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days). Effects on gonadal function, mating behavior, conception, development of the conceptus, parturition, litter size, pup survival, sex, pup body weight and the presence of gross external morphological alterations were assessed. In addition, a gross necropsy and histopathology of the adults was conducted with an emphasis on organs of the reproductive system. Males were dosed via the diet for at least 14 days prior to mating, continuing throughout mating, for 37 days. Females were dosed by dietary exposure for 14 days prior to breeding, and continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days).

Positive control:

no

Parental animals: Observations and examinations:

Daily Observations
A cage-side examination was conducted twice daily, designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals for all males pre-exposure and weekly throughout the study. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14 and 20. Females that delivered litters were subsequently evaluated on LD 0, 1 and 4, and on additional days if warranted by observations made during daily cage-side examinations. Females that failed to mate or deliver a litter were examined weekly. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Body Weights/Body Weight Gains
Body weights for males were recorded on test days -1, 1, 4, 7, and weekly thereafter. Females were weighed on test days 1, 4, 7, and 14 during the pre-breeding period. During gestation, females were weighed on GD 0, 7, 14, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

Feed Consumption
Feed consumption for all animals was measured on test days 1, 4, 7 and 14 during the pre-breeding period by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was measured weekly for males. For mated females, feed consumption was measured on GD 0, 7, 14, and 20. For females delivering litters, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter.

Litter observations:

Litter Data
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of delivery was recorded as the first day the presence of the litter was noted and was designated as LD 0. Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (day 0), the number of live and dead pups on LD 0, 1, and 4, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they are observed during the lactation period. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, to the extent possible, for external and visceral defects and discarded.

Postmortem examinations (parental animals):

Adult Necropsy
A complete necropsy of all the adults was performed. All males were necropsied on test day 38, while females that delivered litters were necropsied on LD 4. Females that did not deliver a litter were necropsied at least 24 days after the last day of the mating period. In all cases, dosing continued until the day prior to sacrifice at which time the animals were fasted overnight. Fasted adult rats submitted alive for necropsy were anesthetized by the inhalation of carbon dioxide, weighed, and their tracheas exposed and clamped. The animals were then euthanized by decapitation.A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The uteri of all females were stained with a 10% solution of sodium sulfide stain for approximately two minutes and were examined for the presence and number of implantation sites (Kopf et al., 1964). After evaluation, the uteri were gently rinsed with saline and preserved in neutral phosphate 10% formalin. Weights of the epididymides, kidneys, liver, and testes were recorded, and organ:body weight ratios calculated.
Histopathology
Tissues with relevant gross lesions was conducted on all adult rats from the control and high-dose groups. The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. Examination of tissues from the remaining groups was limited to the liver (males and females), cervix, ovaries, oviducts, uterus, vagina, and relevant gross lesions. The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. The presence and integrity of the 14 stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change would not be expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not be life threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may be life threatening.The above grading criteria were not sufficiently flexible to accurately characterize the liver microscopic vacuolization consistent with fatty change found in this study. Therefore, the following criteria were applied only to the observation of "liver vacuolization, consistent with fatty change, hepatocyte, multifocal": grade 1 - infrequently observed, grade 2 - occasionally observed, and grade 3 - readily observed.

Postmortem examinations (offspring):

Offspring NecropsyAll pups surviving to LD 4 were euthanized by oral administration of sodium pentobarital solution, examined for gross external alterations, and then discarded. Any pups found dead were examined to the extent possible.

Statistics:

see below

Reproductive indices:

Calculation of Reproductive Indices Reproductive indices were calculated for female and male mating indices, male conception index, female and male fertility indices, gestation index, gestation survival index, post-implantation loss, and days 1 and 4 pup survival indices.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Gestation body weights and body weight gains of females given 1000 mg/kg/day could not be compared due to a lack of viable litters at this dose level.

Dams given 300 mg/kg/day exhibited a non-statistically significant lower (-5.5%) gestation day 20 body weight, corresponding with a statistically significant lower body weight gain between GD 14 and 20 (-40.3%) and non significant over the whole gestation GD 0-20 (-16%). This correlated closely with an approximate 50% reduction in mean litter sizes observed in this group. Therefore it was not due to parental toxicity.

No treatment-related effects at 100 mg/kg/day or during the lactation phase of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, hepatocellular changes were noted in all animals: very slight diffuse cytoplasmic microvacuolization of periportal hepatocytes and multifocal hepatocyte vacuolization consistent with fatty change, grading from "infrequently observed" to "readily observed". Similar effects were seen in nearly all treated females at 100 and 300 mg/kg/day with similar severity. Since there was no evidence of increased hepatocellular necrosis, all above effects were interpreted as reversible.

At 300 and 1000 mg/kg/day, observation of pigment laden macrophages within the endometrial stroma (multifocal, very slight) was consistent with embryonic resorption.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

embryo-fetal effects:

At 300 and 1000 mg/kg/day, observation of pigment laden macrophages within the endometrial stroma (multifocal, very slight) was consistent with embryonic resorption.

At 300 and 1000 mg/kg/day: marked increase in post- implantation loss (embryonic resorption, 70% and 100%, resp.). High incidence of complete litter resorption (4/12 and 12/12 pregnant females, resp.; no litters produced at 1000 mg/kg/day).
At 300 mg/kg/day, the remaining 8/12 pregnant females showed partial litter losses to varying degrees, leading to statistically lower mean number of live pups at birth, and days 1 and 4 postpartum (all ~-47%), consistent with the increase in post-implantation loss.
It is estimated that the embryonic resorptions occurred sometime toward the end of the second week of gestation (implantation is completed on GD 6; full term is 21-22 days), with some of the high dose animals perhaps occurring slightly earlier. In the 100 mg/kg/day group, all reproductive parameters were comparable to control values.

There were no treatment-related embryo-fetal effects at 100 mg/kg/day. There were no treatment-related effects at any dose level on mating, conception, fertility, time to mating, gestation length.

Key result

Dose descriptor:

NOAEL

Remarks:

parental/systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 213 mg/kg bw/day AMP

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

parental/systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 71 mg/kg bw/day AMP

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 71 mg/kg bw/day AMP

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

reproductive performance

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 213 mg/kg bw/day AMP

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 710 mg/kg bw/day AMP

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

no pup mortality after delivery

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup body weights in the 300 mg/kg/day group were increased on days 1 and 4 postpartum, with the male mean pup weights identified as statistically different. These increases were most likely due to the decreased litter size at this dose level, as pup body weight varies inversely with litter size (Agnish and Keller, 1997). This was non-adverse.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No effect on pup sex ratio or external morphologic alterations at any dose.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Very few examinations are performed since offspring is only examined until day 4. Effects on fetal toxicity are reported under "reproductive performance" in Parental animals.

Key result

Generation:

F1

Remarks on result:

not determinable because of methodological limitations

Remarks:

An OECD 421 study does not allow to determine any NOAEL in F1 generation since pups are only examined until day 4. Pups did not show any adverse effects until day 4. Embryo-fetal toxicity is covered by the Reproductive NOAEL under Parental generation.

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

F0 / Incidence of treatment-related Liver Effects

Sex	Male				Female
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
Liver (# examined)	12	12	12	12	12	12	12	12
Vacuolization, cytoplasmic, hepatocyte, periportal, diffuse, very slight	2	2	3	12	3	12	11	12
Vacuolization, consistent with fatty change, hepatocyte, multifocal:	2	2	1	10	3	12	11	11
infrequently observed	2	2	1	3	3	5	6	5
occasionally observed	0	0	0	6	0	3	1	5
readily observed	0	0	0	1	0	4	4	1

Bold type indicates the effects judged to be treatment related.

 

F0 / Incidence of post-implantation loss

Sex	Female
Dose (mg/kg/day)	0	100	300	1000
Postimplantation Loss (%)	9.69	6.69	69.72	100.0

Conclusions:

In a rat dietary reproductive screening study (much less robust than the OECD 443 study) with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP induced post-implantation loss from 213 mg AMP/kg bw/day, with a reproduction/development NOAEL of 71 mg AMP/kg bw/day. The NOAEL for parental toxicity was 213 mg AMP/kg bw/day in males and < 71 mg AMP/kg bw/day in females.

Executive summary:

In an OECD 421 reproductive toxicity screening study, groups of 12 CD rats/sex/dose were fed diets supplying 0 (control), 100, 300, or 1000 mg/kg bw/day AMP-HCl (containing 71, 213 and 710 mg AMP/kg bw/day, resp.). Males were exposed for at least two weeks prior to breeding and throughout breeding for 37 days. Females were exposed for two weeks prior to breeding, throughout breeding (up to two weeks), gestation (three weeks) and lactation (four days). Resulting duration of treatment: F0 males: 5 weeks, F0 females: <8 weeks, F1: <4 weeks (indirect via gestation and 4-day lactation). The following effects were noted:

• At 710 mg AMP/kg bw/day, liver weights were increased in males only. Hepatocellular changes were noted in all animals: very slight diffuse cytoplasmic microvacuolization in periportal zone and multifocal vacuolization consistent with fatty change (grade: up to "readily observed"). Kidney weights were increased in males, non toxicologically relevant in absence of histopathological changes. Treatment caused total post-implantation loss (embryo resorption) with complete litter loss in all 12 females, correlated with pigment laden macrophages in endometrial stroma. Gestation body weight (gains) of females could not be interpreted due to lack of viable litters.


• At 213 mg AMP/kg bw/day, females showed similar hepatocellular changes as at 710 mg AMP/kg bw/day. Post-implantation loss was increased to 70% (vs. 10% in controls) due to litter loss (4/12 complete, 8/12 partial), resulting in lower number of live pups, increased pup body weight, decreased maternal gestation body weight (gain) and pigment laden macrophages within maternal endometrial stroma.


• At 71 mg AMP/kg bw/day, females showed similar hepatocellular changes as at 213 and 710 mg AMP/kg bw/day. There was no effect on reproductive performance.

AMP-HCl had no effect on mating performance or embryo/pup development at any dose-level. Based on liver changes, the no-observed effect level (NOEL) for parental toxicity was 213 mg AMP/kg bw/day in males and < 71 mg AMP/kg bw/day in females. The NOEL for reproductive effects was 71 mg AMP/kg bw/day in females due to post-implantation loss and >=710 mg AMP/kg bw/day in males.

Below conclusions were drawn post-report by the registrant:

1) As fatty liver was "readily observed" in some animals at the high- (both sexes), mid- and low-dose levels (females only), it is proposed to consider by default this level of fatty change as adverse and to set NOAELs at same values as corresponding NOELs in this study.

2) Compared to the OECD 443 study in same strain of rats and same dietary administration method, this study is much less robust as it includes half less animals (12 vs. 19-30/sex/dose-level) and exposure period is much shorter (F0 males: 5 weeks, F0 females: <8 weeks, F1: < 4 weeks indirect via gestation/4-day lactation; vs. in OECD 443: F0 males/females: 18 weeks, F1: 6 weeks indirect via gestation/lactation + 13-14 weeks via diet). Also, three OECD 414 studies are available for robust information on development.

3) At the LOAEL for general and reproductive toxicity (females: 71 and 213 mg AMP/kg bw/day, resp.), diet contained 0.123-0.164 and 0.307-0.369 % w/w AMP-HCl, resp. (see report p. 38) i.e. 0.087 to 0.262% w/w AMP. AMP was tested neutralized as AMP-HCl, CAS No. 3207-12-3. As is, AMP is alkaline (high-purity-grade: pKa = 9.70). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3189 ln(Concentration in % w/w) + 11.401], pH of high-purity-grade AMP solutions at 0.087-0.262 % AMP w/w would range 10.6-11.0. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is instead of AMP-HCl, the study's LOAEL for general and reproductive toxicity could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines referenced by REACH do NOT require any neutralization or pH adjustment of test items, this study represents artificial dose maximization in excess of REACH principles, removing AMP's critical toxic effect which is pH-dependent non-specific toxicity.

4) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

142 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

very high

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Influence of pH adjustment on relevance of toxicology studies for hazard classification (see pH and pKa, IUCLID § 4.20/4.21):

Bibliography on maximum tolerated pH, together with pH/concentration curves for solutions of industrial and high-purity AMP grades (Fernandes, 2023), allow to conclude that repeated administration of non-neutralized AMP solutions above:

• 0.0005% w/w for oral route (reaching pH 9, maximum tolerated pH by oral route according to Turner, 2011), or


• 0.8% w/w for topical application (reaching pH 11.5, maximum tolerated pH for skin according to Shu-Hua 2020, REACH regulation 2006, ECHA 2017),

would lead to animal death or severe dose-limiting suffering. In almost all AMP in vivo toxicology studies (see IUCLID § 7.5.1, 7.5.3, 7.8), neutralization of AMP (pH adjustment or testing as AMP-HCl) allowed to test much higher concentrations and (in some studies) observe toxic effects. Since OECD guidelines do NOT require any neutralization or pH adjustment, such studies represent artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity.

This is confirmed experimentally by the 4 in vivo repeat-dose studies (see IUCLID §7.5.1 and 7.5.2) where AMP was tested without pH adjustment (there is no reproductive toxicity study without pH adjustment):

• A 13-week oral rat study (Pittz 1977/79) was done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralization to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralizing AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.


• In a 5-day study on rats inhaling non-neutralized AMP aerosols (Sullivan 2017), local corrosion to skin and respiratory tract occurred at lower airborne concentrations (LOAEC: 700 mg/m3) than liver toxicity (NOEC: 700 mg/m3, LOEC: 1400 mg/m3, minimal grade liver vacuolation). pH of the 10% aerosol was calculated as 12.3 using the pH curves. Thus, protecting against local effects upon inhalation will protect against systemic effects.


• In 5-day oral studies in rats and monkeys (Pittz 1977) where non-neutralized AMP reached pH >11, mortality occurred in both species. The maximum tolerated dose (MTD) in mg AMP/kg bw/day were: well below 500 in female monkeys; 500 in female rats; 1000 in male rats. No NOAEL could be set in either species due to a 10-day recovery before necropsy and liver examinations.

 

Ingestion (only route with reproductive effects and adverse liver toxicity) is a negligible exposure route for REACH identified uses:

Reproductive effects were only observed after direct ingestion of AMP (see §7.8.1, 7.8.3), and not in two dermal studies (see §7.8.2). In humans, direct ingestion only happens via the OTC drug pamabrom and such US pharma uses are out of scope of REACH/GHS/CLP. AMP is not an approved food additive in EU and is not used to treat drinking water so REACH identified uses do not involve direct ingestion of AMP. AMP is extremely water soluble, readily biodegradable and non-bioaccumulative so no significant oral exposure to AMP via environmental emissions is expected from REACH identified uses. For all these reasons, toxicology data acquired by oral route are an extreme, barely realistic worst-case of actual systemic toxicity potential by exposure routes related to REACH uses. Indeed, by skin contact and inhalation, systemic exposure potential to AMP is very limited due to:

• risk management measures protecting against local corrosive effects of AMP and its industrial formulations (see IUCLID § 4.20/4.21, pH/pKa and also CSR);


• negligible volatility (see IUCLID § 4.6 Vapour pressure: 45 Pa at 20°C);


• few identified uses with a potential to generate aerosols (see IUCLID § 3.5 Use information);


• low concentration in end products (use as pH modifier, see CSR);


• only 7-17% dermal absorption through human skin (see IUCLID § 7.1.2 Dermal absorption).

Effects on developmental toxicity

Description of key information

Animal development data on AMP after pH adjustment (see Developmental toxicity /IUCLID § 7.8.2):

Three GLP and OECD 414 guideline-compliant studies are available, where pregnant rats or rabbits were treated orally or dermally with AMP after pH adjustment. Results are summarized below.

Table 2: Overview of animal developmental studies with pH-adjusted AMP

Reference	Study design		Developmental effects			Maternal/general toxicity
Author year	Test item	Species, route	Adverse effects; LOAEL	NOAEL	AMP concentration in dosage form at LOAEL; corresponding pH if not neutralized	Adverse effects; LOAEL	NOAEL	AMP concentration in dosage form at LOAEL; corresponding pH if not neutralized
Murphy 2020	AMP neutralized to pH 6.6-7.5	Rabbit, oral gavage	Effects secondary to extreme maternal toxicity above MTD (one survivor: fewer implantation sites, preimplantation loss, slightly lower fetal and litter weights, less live fetuses per litter; decedents: total litter loss, dead fetuses); 75	25	1.42-1.46 % w/w; 11.5	MTD largely exceeded notably with 19/20 deaths and body weight loss (-17%) due to anorexia (-73% consumption). Adverse liver lesions (minimal to moderate liver degeneration, necrosis and hepatocellular vacuolation, minimal liver ductular hyperplasia); 75	25	1.42-1.46 % w/w; 11.5
Murphy 2020	AMP neutralized to pH 6.7-7.5	Rat, oral gavage	none; N/A	300	N/A	14% increased liver weight, minimal to mild hepatocellular vacuolation in 23/25 animals, minimal liver necrosis in 3/25 animals; 100	30	1.83-1.84 % w/w; 11.5
Carney 2006	AMP adjusted to pH 9.5	Rat, dermal 6h/day	none; N/A	300	N/A	Local: scaling and fissuring of skin (slight: 24/26; moderate to severe: 9/26), scabs (20/26 animals); 300 Systemic: none; N/A	Local: 100 Systemic: 300	34.2 % w/w; 12.1

Bold: key studies; all NOAEL/LOAEL values in mg AMP/kg bw/day; M: males; F: females; N/A: not applicable; red: excess pH, MTD exceeded if AMP hadn't been neutralized

The two oral studies are key studies as they allow to conclude on systemic effects. The dermal study is supportive: in spite of pH adjustment to 9.5, the alkaline material breached the skin barrier at the top-dose, leading to supra-linear systemic exposure. However, this dermal study confirms that no adverse developmental effects are expected for uses involving skin contact. Also, it shows that liver toxicity occurs after oral, but not dermal, exposure.

In the two rat studies, AMP had no effect on development once the embryo implanted (treatment started on gestation day 6). The rabbit top-dose was too high vs. guideline recommendations, leading to non-specific developmental effects secondary to animal death (all but one female), bad clinical condition, weight loss and anorexia. At the rabbit mid-dose, no adverse effect occurred once the embryo implanted (treatment started on gestation day 7 in rabbits). All three studies identified a clear threshold (NOAEL) for developmental effects.

All adverse effects observed in the three OECD 414 studies occurred at dose-levels (LOAELs) which cannot be reached with AMP (base) as dosage form pH would have ranged 11.5-12.1 (see red values in Table 2), compared to a tolerable maximum of 9 by oral route (Turner 2011, see IUCLID section 4.20) and a cut-off of 11.5 above which ECHA recommends to consider a substance as Skin Corrosive cat 1 and waive all dermal application studies. The systemic LOAELs of 75 and 100 mg AMP/kg bw/day in rabbit and rat oral studies could only be reached thanks to pH neutralization of dosage forms. This represents artificial, supra-guideline dose maximization as test item neutralization is not required by OECD guidelines.

The rat oral study showed that liver toxicity occurs at dose-levels without developmental effects, confirming the same pattern as for effects on fertility (post-implantation loss). Mechanistic studies (summarized under "Mode of Action Analysis / Human Relevance Framework" below) investigating liver toxicity (hepatocyte vacuolation) and reproductive effects (focus on post-implantation loss) suggested that these two effects share a common pathway: they are both associated with or correlated to, perturbation of choline uptake and choline metabolism into phospholipids.

Taken together, these facts and considerations allow to conclude that no developmental toxicity classification is warranted for AMP (CAS N° 124-68-5). This is further detailed below under "Justification for classification or non-classification‌".

 

Human data on pamabrom (see Other studies /IUCLID § 7.8.3 and Exposure related observations in humans, IUCLID § 7.10.3):

Pamabrom is an Over The Counter (OTC) diuretic agent which is an AMP salt of 8-bromotheophylline, CAS No. 606-04-2.
It is an equimolar mixture of 74.4% w/w 8-Bromotheophylline (CAS No. 10381-75-6) and 25.6% w/w 2-amino-2-methylpropan-1-ol (AMP, CAS No. 124-68-5). Clinical data on this test material can be used for AMP safety assessment based on the AMP/Pamabrom bioequivalence study (see Basic Toxicokinetics /IUCLID §7.1.1) which indicated that Pamabrom's AMP, and AMP as is, were equivalent in terms of AUC0-t, AUC0-inf and AUC0-168h. Pamabrom doses can be converted into equivalent AMP doses based on the AMP content of 25.6% w/w in this drug and assuming a patient weight of 70 kg when not indicated.

A) Oral clinical studies:

Six clinical studies are available on pamabrom, two of which included pregnant women. NOAELs are expressed in AMP based on pamabrom posology and AMP content. In all cases the below-listed NOAELs were the maximum tested dose:

1) Patterson, 1958: No adverse effects in n=38 pregnant women with edema classified as mild pre-eclampsia. The study notably monitored gravidity, gestation, changes in fetal movement. The NOAEL was >= 5.9 mg AMP/kg bw/day x 1 week (second treatment cycle), and >= 2.9 mg AMP/kg bw/day x 2 weeks (both treatment cycles in patients treated twice).

2) James et al, 1957: No adverse effects in n=180 pregnant women with edema treated for unknown duration (Klimisch 4 study). The max. NOAEL was >= 2.2 mg AMP/kg bw/day, and in most patients it was >=1.6 mg AMP/kg bw/day.

B) 70 years of safe clinical use - Pharmacovigilance argument:

In the US, pamabrom is used since early 1950's so it has a track-record of >70 years of safe use in an ill population. Based on the National Library of Medicines database, at least 20 US OTC drugs currently contain pamabrom (full list provided in study summary). Their claims include back/leg/joint pain and edema (1 drug which may thus be used during pregnancy) and premenstrual and menstrual pain/discomfort/edema (19 drugs which may thus be used in women of child-bearing age before pregnancy). The only mention related to reproduction is a generic statement used for almost all drugs: "If pregnant or breast-feeding, ask a health professional before use." Based on contents and posology for each drug, AMP has a human NOAEL of >=0.73 mg AMP/kg/day for up to 10-day treatment cycles, repeated over periods.

C) Overall human oral NOAEL for reproduction:

No adverse reproductive effect was ever reported during 70 years of clinical trials and medication with pamabrom based on 5 publications (6 trials) and posology of 20 OTC drugs. The most robust human oral NOAEL covering reproduction comes from Patterson, 1958: >= 5.9 mg AMP/kg bw/day x 1 week and >= 2.9 mg AMP/kg bw/day x 2 weeks. All other studies and OTC drug posology confirm AMP NOAEL values in the mg/kg bw/day range, always the highest tested dose whatever the treatment duration (3 days to 17 weeks + one trial where treatment duration was not indicated). To note, the 2-week reproductive NOAEL is the same value as the human 4-week repeat-dose toxicity NOAEL (see IUCLID § 7.5), although it does not come from the same publication, authors, year and target population. This shows that at realistic human exposure doses, AMP has neither repeat-dose nor reproductive toxic effects even when ingested daily.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

AMP-95: industrial grade

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Text Table 3: Animal Information
Females
Species and Strain Time-mated female New Zealand White Rabbits
Supplier Charles River Breeding Labs; Saint Constant, Canada
Method of Identification Cage Card/Tattoo
Number of Animals Received 82
Number Used on Study 80
Age at First Dose 4.6 – 6.2 months
Weight Range at Mating 3245 – 4499 g
Weight Range at First Dose 3164.9 – 4352.1 g

Animals were acclimated to laboratory conditions for at least three days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance, AMP-95, was supplied as an 882 mg AMP/mL solution with an expiration date of 06 April 2024. The test substance was received at ambient temperature and stored at room temperature upon receipt. The control substance, DI water, was received from a vendor/supplier and stored at room temperature upon receipt. Certificates of Analysis are presented in Appendix 1. Reserve samples (1 mL) of the neat test substance and control substance were taken and subsequently archived under the same storage conditions as those of the neat materials.
The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control article, DI water, was used as received and adjusted for pH using NaOH or HCl on each day of dosing. Final pH ranged from 6.96-7.42. Formulations were stored at room temperature until used for dosing.
Group 2 (6 mg/mL), Group 3 (20 mg/mL), and Group 4 (60 mg/mL) formulations were prepared daily, with the exception of the last preparation, by adding the required amount of test substance to the appropriate amount of the vehicle/control substance and mixing until visually clear. Formulations were adjusted for pH using 1N NaOH and/or HCl. The final pH ranged from 6.64-7.47. Additional vehicle was added until the required final volume was reached. Formulations were kept at room temperature until used for dosing on the day of preparation. The last preparation was assigned a 7-day shelf life and stored at room temperature until used for dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were sampled and stored.
Stability analysis of the dose formulations was performed under Inotiv Study No. PRO.1000-192139-001 as part of the method validation.
One set of samples was shipped (on dry ice) to the Inotiv West Lafayette for test substance concentration analysis. Following receipt of analytical results, unused samples were discarded with authorization from the Study Director.

Duration of treatment / exposure:

The animals were dosed once daily from GD 7 to 28 (day of confirmation of mating = GD 0) via oral gavage at a volume of 5 mL/kg. Dose volumes were based on the most recent body weights.

Frequency of treatment:

Once daily

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

contains 9.5 mg/kg bw/day AMP, taking into account 5.4% water content

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

contains 23.7 mg/kg bw/day AMP, taking into account 5.4% water content

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

contains 71.0 mg/kg bw/day AMP, taking into account 5.4% water content

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2 or more then 2 times daily
- Cage side observations checked in table [Yes] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On presumed GD 29

BODY WEIGHT: Yes
- Time schedule for examinations:GD 7, 10, 13, 16, 19, 21, 24, and 27
Prior to necropsy (GD 29)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined:
Esophagus,Trachea,Lungs,Uterus,parathyroid & thyroid gland, spleen,stomach, thymus,Kidney, Intestine,Ovary,Pancreas,mammary gland, heart and liver
OTHER: liver weight, gravid uterine weight

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
Number of late resorptions: No
- Other:

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: No data

Statistics:

ANOVA
Shapiro-Wilk test
Levene’s test
Dunnett’s t-test

Indices:

Percent Pre-implantation Loss = [(total number of corpora lutea – total number of implantations) / total
number of corpora lutea] X 100
Percent Post-implantation Loss = (total post-implantation loss / total number of implantations) X 100

Historical control data:

Persistent truncus arteriosus has been noted in a larger developmental and reproductive historical control data base from Charles River Ashland (mean 0.03 % per litter, maximum 0.7% per litter), and has been observed as a spontaneous malformation in rabbits (Hood, 2012). It is considered to be a heritable trait and the genetic makeup of both parents could influence the penetrance and/or spontaneous observation (Hood, 2012). Due to the low incidence and absence of any other evidence of maternal or developmental toxicity at
≤ 25 mg/kg/day, this malformation was not considered test substance related.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at 75 mg/kg/day: adverse clinical observations of red/bloody discharge (from vagina, nose), few feces, pale, audible respiration, and languid (hypoactivity)

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

at 75 mg/kg/day: only one rabbit survived until schedule termination; all other rabbits were euthanized as moribund (17/20) or found dead (2/20).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at 75 mg/kg/day: body weight loss (mean body weights 0.93x controls on GD 21)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at 75 mg/kg/day: decreased food consumption (approximately 30 grams vs. 110 in controls on GD 20-21)

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One female at 25 mg/kg/day and 11 females at 75 mg/kg/day had discolored liver, described as pale tan, or tan.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

75 mg/kg/day: minimal to moderate liver vacuolar degeneration/necrosis + minimal to moderate liver hepatocellular periportal macrovesicular vacuolation + mild to moderate multifocal liver necrosis + minimal liver ductular hyperplasia
25 mg/kg/day: minimal to mild liver hepatocellular periportal macrovesicular vacuolation + minimal ductular hyperplasia
10 mg/kg/day: minimal liver hepatocellular periportal macrovesicular vacuolation in 1/20 animals only

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

The top-dose of 75 mg/kg/day largely exceeded the MTD based notably on mortality in 19/20 rabbits. Therefore effects in this group cannot be used to assess reproductive and developmental effects.

Number of abortions:

not specified

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination and therefore the effect of AMP-95 on fetal survival (abortions) could not be assessed on GD 29.
No effect at lower doses.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. In this animal there were fewer implantation sites (8 vs. 10.9 in controls), with 27% pre-implantation loss and 0% post-implantation loss.
No effect at lower doses.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. At unscheduled necropsies, there were six rabbits with total litter loss consisting of primarily late resorptions (compared to 0 in controls). This was due to low food consumption.
No effect at lower doses.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. At unscheduled necropsies, there were
- 6 rabbits with total litter loss consisting of primarily late resorptions (compared to 0 in controls)
- only 1 rabbit with live fetuses
This was due to low food consumption.
No effect at lower doses.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. At unscheduled necropsies, there were nine dead fetuses in three litters (compared to 0 in controls). This was due to low food consumption.
No effect at lower doses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The top-dose of 75 mg/kg/day largely exceeded the MTD based notably on mortality in 19/20 rabbits. Therefore effects in this group cannot be used to assess reproductive and developmental effects.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 23.7 mg/kg bw/day AMP, taking into account 5.4% water content

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

pre and post implantation loss

total litter losses by resorption

Key result

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 71.0 mg/kg bw/day AMP, taking into account 5.4% water content

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

not specified

Description (incidence and severity):

75 mg/kg/day: Slightly lower mean fetal weight (34.9 g vs. 38.8 g in controls); statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses)
No effect at lower doses

Reduction in number of live offspring:

not specified

Description (incidence and severity):

75 mg/kg/day: Slightly lower number of live fetuses per litter (8 vs. mean of 10.8 in controls); statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses)
No effect at lower doses

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Description (incidence and severity):

75 mg/kg/day: Slightly lower litter weight (279.3 g vs. mean of 412.8 g in controls); statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses)
No effect at lower doses

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

The top-dose of 75 mg/kg/day largely exceeded the MTD based notably on mortality in 19/20 rabbits. Therefore effects in this group cannot be used to assess reproductive and developmental effects. There were some minimal growth/survival effects at the top-dose (lower fetal and litter weights, less live fetus) but statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses); effects would be attributable to the excess maternal toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 23.7 mg/kg bw/day AMP, taking into account 5.4% water content

Sex:

male/female

Remarks on result:

other: the next higher dose resulted in so high maternal toxicity that the relevance of the growth/survival effects observed (lower fetal and litter weight, less live fetuses) cannot be judged

Remarks:

maternal no observed adverse-effect level (NOAEL) was determined to be 25 mg/kg/day and the fetal NOAEL was determined to be 25 mg/kg/day.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a rabbit oral developmental toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, 71.0 mg AMP/kg bw/day led to excess maternal toxicity triggering a cascade of non-specific developmental effects.
The NOAEL was 23.7 mg AMP/kg bw/day for maternal and developmental effects.

Executive summary:

In an OECD 414 study, pregnant New Zealand White rabbits were orally gavaged with AMP-95 (neutralized to pH 6.6-7.5) at dose levels of 0, 10, 25 or 75 mg/kg bw/day over GD 7-28 (containing 9.5, 23.7 and 71.0 mg AMP/kg bw/day resp., taking into account 5.4% water content). Surviving does were subjected to a gross necropsy,and uterine and fetal examinations on presumed GD 29.

• 71.0 mg AMP/kg bw/day largely exceeded the MTD. Mortality occured in 19/20 rabbits. Red/bloody discharge (from vagina, nose), few feces, pale, audible respiration, and languid (hypoactivity) were observed. Body weight loss occured (-17%) due to markedly reduced food consumption (-73% on GD 20). 11 rabbits had discolored liver, and there were multiple adverse liver lesions (minimal to moderate liver vacuolar degeneration/necrosis + minimal to moderate liver hepatocellular periportal macrovesicular vacuolation + mild to moderate multifocal liver necrosis + minimal liver ductular hyperplasia). The surviving rabbit had fewer implantation sites (8 vs. 10.9 in controls), with 27% pre-implantation loss and 0% post-implantation loss. Several decedent rabbits showed total litter loss consisting of primarily late resorptions and dead fetuses were noted. Mean fetal weight, litter weight and number of live fetuses per litter were slightly lower in the litter from the surviving rabbit. Statistical and biological significance of developmental effects cannot be determined, but they can be considered seconadry to the very high maternal toxicity.


• At 23.7 mg AMP/kg bw/day, one rabbit had discolored liver, and liver lesions were non-adverse (minimal to mild liver hepatocellular periportal macrovesicular vacuolation + minimal ductular hyperplasia). No developmental effects.


• At 9.5 mg AMP/kg bw/day, liver lesions were non-adverse (minimal liver hepatocellular periportal macrovesicular vacuolation in 1 animal only). No developmental effects.

Under the conditions of this study, the NOAEL for maternal toxicity was 23.7 mg/kg bw/day. The NOAEL for developmental toxicity was 23.7 mg/kg bw/day, the highest dose level without excess maternal toxicity.

Below conclusions were drawn post-report by the registrant:

1) At the toxic dose of 71.0 mg AMP/kg bw/day, AMP concentration in dosage form ranged 14.24-14.61 mg/mL (see report p. 124) i.e. 1.42-1.46 % w/w AMP. AMP was tested neutralized to pH 6.6-7.5 using HCl. As is, AMP is alkaline (industrial-grade: pKa = 9.74). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3309 ln(Concentration in % w/w) + 11.548], pH of industrial-grade AMP solutions at 1.42-1.46 % AMP w/w would be 11.7. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is, the study's toxic dose could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines do NOT require any neutralization or pH adjustment, this study represents artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity.

2) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

AMP-95: industrial grade

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Text Table 3: Animal Information
Females
Species and Strain Time-mated female Sprague Dawley Rats
Supplier Charles River Breeding Labs; Raleigh, NC
Method of Identification Cage Card/Ear Tag
Number of Animals Received 105
Number Used on Study 100
Age at First Dose 9 – 12 weeks
Weight Range at Mating 180 – 223 g
Weight Range at First Dose 202.0 – 260.6 g
Disposition of Extra Animals Euthanized
Animals were acclimated to laboratory conditions for at least two days prior to the first dose and released
from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number
that was recorded on each cage label.
14.2.2 Husbandry

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance, AMP-95, was supplied as an 882 mg AMP/mL solution with an expiration date of
06 April 2024. The test substance was received at ambient temperature and stored at room temperature
upon receipt. The control substance, DI water, was received from a vendor/supplier and stored at room
temperature upon receipt. Certificates of Analysis are presented in Appendix 1.
Reserve samples (1 mL) of the neat test substance and control substance were taken and subsequently
archived under the same storage conditions as those of the neat materials.
All remaining test and control substance were retained for use on subsequent studies. All remaining
vehicle/control substance components were retained for use on subsequent studies. All empty containers
were discarded after completion of the in-life phase of study.The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control article, DI water, was used as received and adjusted for pH using NaOH or HCl on each
day of dosing. Final pH ranged from 6.96-7.42. Formulations were stored at room temperature until used
for dosing.
Group 2 (6 mg/mL), Group 3 (20 mg/mL), and Group 4 (60 mg/mL) formulations were prepared daily, with
the exception of the last preparation, by adding the required amount of test substance to the appropriate
amount of the vehicle/control substance and mixing until visually clear. Formulations were adjusted for pH
using 1N NaOH and/or HCl. The final pH ranged from 6.72-7.46. Additional vehicle was added until the
required final volume was reached. Formulations were kept at room temperature until used for dosing on
the day of preparation. The last preparation was assigned a 7-day shelf life and stored at room temperature
until used for dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control article, DI water, was used as received and adjusted for pH using NaOH or HCl on each
day of dosing. Final pH ranged from 6.96-7.42. Formulations were stored at room temperature until used
for dosing.
Group 2 (6 mg/mL), Group 3 (20 mg/mL), and Group 4 (60 mg/mL) formulations were prepared daily, with
the exception of the last preparation, by adding the required amount of test substance to the appropriate
amount of the vehicle/control substance and mixing until visually clear. Formulations were adjusted for pH
using 1N NaOH and/or HCl. The final pH ranged from 6.72-7.46. Additional vehicle was added until the
required final volume was reached. Formulations were kept at room temperature until used for dosing on
the day of preparation. The last preparation was assigned a 7-day shelf life and stored at room temperature
until used for dosing.
Stability analysis of the dose formulations was performed under Inotiv Study No. 1000-192139-001 as part
of the method validation; the methodology is presented in Appendix 2.
One set of samples was shipped (on dry ice) to the Inotiv West Lafayette for test substance concentration
analysis; the methodology is presented in Appendix 3. Following receipt of analytical results, unused
samples were discarded with authorization from the Study Director.

Duration of treatment / exposure:

The animals were dosed once daily from GD 6 to 20 (day of confirmation of mating = GD 0) via oral gavage at a volume of 5 mL/kg. Dose volumes were based on the most recent body weights.

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Remarks:

contains 28.4 mg/kg bw/day AMP, taking into account 5.4% water content

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

contains 94.6 mg/kg bw/day AMP, taking into account 5.4% water content

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

contains 284 mg/kg bw/day AMP, taking into account 5.4% water content

No. of animals per sex per dose:

25 mated Female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results from previous toxicology studies performed by the Sponsor.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: >2 time daily
DETAILED CLINICAL OBSERVATIONS:NO
BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded on GD 0, 6,9, 12, 15, 18 and prior to necropsy (GD 21).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was calculated for each interval, including GD 6-9, 9-12, 12-15, 15-18

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): NO

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Liver

OTHER:
Hormone Analysis (T3,T4 and TSH)
Gross Necropsy, Liver Weights
Uterine Examination
Fetal Examinations
Histopathology

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

Blood samples were collected from all dams (not fasted) prior to termination on GD 21 for thyroid
hormone analysis.

Fetal examinations:

All live fetuses were individually weighed, identified, sexed, and examined for external malformations and variations. Following completion of the external examination of all fetuses in the litter, each fetus was euthanized via an oral dose of Euthasol® solution (0.03 mL/fetus).
Approximately one-half of the fetuses in each litter were examined viscerally by fresh tissue dissection. The internal sex was recorded. The heads were preserved in Bouin’s and examined by Wilson’s sectioning. The remaining fetuses had the internal sex recorded and then were eviscerated, preserved, stained with Alizarin Red S, and examined for skeletal abnormalities. Fetal findings were classified as malformations or developmental variations.

Statistics:

Electronic data collection, including randomization, dose formulations and dispensing, dosing, animal husbandry, environmental enrichment, physical examinations, cageside observations, body weights, body weight changes, food consumption, liver weights, uterine and pregnancy data, gross pathology, fetal examination data, and statistical analysis was performed using Provantis™ Version 8 (Instem LSS, Limited; Stone, UK). Environmental monitoring was performed using Rees Environmental Monitoring System (Rees Scientific Corporation; Trenton, NJ).
Quantitative data (body weights, body weight changes, food consumption, and liver weights) from the treated groups were compared statistically to the data of the control group using one-way Analysis of Variance (ANOVA) techniques.
Some quantitative data (body weight changes adjusted for gravid uterine weight, thyroid hormone levels, and uterine data) were analyzed using the Kolmogorov-Smirnov test for normality, the Levene Median test for equal variance, and by one-way Analysis of Variance (ANOVA). If either the normality or equal variance test failed, then the analysis was continued using the non-parametric Kruskal-Wallis ANOVA on rank transformed data. For parametric data, if the ANOVA indicated statistical significance among experimental groups then the Dunnett’s t-test was used to delineate which groups (if any) differed from the control.

Indices:

Parameters evaluated during the study included mortality, physical examinations, cage side observations, body weights, body weight changes, food consumption, gross pathology findings, absolute and relative liver weights, uterine data, fetal weights, fetal examination data (external, visceral, skeletal, and head exams), liver histopathology findings, and hormone analysis
Adjusted Body Weight Change = Total Body Weight Change (GD 6 to 21) – Uterine Weight
Percent Pre-implantation Loss = [(total number of corpora lutea – total number of implantations) / total
number of corpora lutea] X 100
Percent Post-implantation Loss = (total post-implantation loss / total number of implantations) X 100
Percent Viable Implantations = (total number of live fetuses / total number of implantations) X 100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute liver weight was 14% higher in females administered 300 mg/kg/day than in controls, without a notable change in mean liver-to-body weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One female administered 300 mg/kg/day (animal 31508) and one female administered 100 mg/kg/day (animal 31491) had macroscopic findings of discolored liver, described as pale/tan or tan, respectively.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

AMP-95-related minimal to mild hepatocellular vacuolation occurred at all dose levels. Incidence and grade of vacuolation were dose-related. However, without associated hepatocyte degeneration or metabolic disturbances, this minimal to mild vacuolation was non-adverse.

Minimal focal or multifocal liver necrosis was observed in 3/25 animals administered 100 and 300 mg/kg/day. It was considered as adverse.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 28.4 mg/kg bw/day AMP, taking into account 5.4% water content

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 94.6 mg/kg bw/day AMP, taking into account 5.4% water content

Basis for effect level:

histopathology: non-neoplastic

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

No effects on development.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a rat oral developmental toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP had no effect on development up to 284 mg AMP/kg bw/day.

Executive summary:

In an OECD 414 study, pregnant Sprague Dawley rats were orally gavaged with AMP (neutralized to pH 6.7-7.5) at dose levels of 0, 30, 100 or 300 mg/kg bw/day (containing 28.4, 94.6 and 284 mg AMP/kg bw/day resp., taking into account 5.4% water content) over GD 6-20.

Dams were subjected to gross necropsy, and uterine and fetal examinations on GD 21. Blood samples were collected from all dams (not fasted) prior to termination on GD 21 for thyroid hormone analysis. Parameters evaluated during the study included mortality, physical examinations, cage side observations, body weights, body weight changes, food consumption, gross pathology findings, absolute and relative liver weights, uterine data, fetal weights, fetal examination data (external, visceral, skeletal, and head exams), liver histopathology findings, and thyroid hormone levels (T3, T4, and TSH).

• 284 mg/kg bw/day produced adverse liver toxicity: mean absolute liver weight was 14% higher in females, one female had discolored liver, minimal to mild (non-adverse) periportal to midzonal macrovesicular hepatocellular vacuolation was observed in 23/25 animals, and minimal focal or multifocal liver necrosis (adverse) was observed in 3/25 animals. There were no effects on development.


• 94.6 mg/kg bw/day produced very similar adverse liver toxicity, except for absence of noteworthy liver weight change.


• 28.4 mg/kg bw/day only produced non-adverse liver changes: minimal to mild hepatocellular vacuolation in 10/25 animals.

Under the conditions of this study, the NOAEL for maternal toxicity based on liver toxicity was 28.4 mg AMP/kg bw/day. The NOAEL for developmental toxicity was 284 mg AMP/kg bw/day, the highest dose level tested.

Below conclusions were drawn post-report by the registrant:

1) At the maternal LOAEL of 94.6 mg AMP/kg bw/day, AMP concentration in dosage forms ranged 18.27-18.36 mg/mL (see report p. 116) i.e. 1.83 % w/w AMP. AMP was tested neutralized to pH 6.7-7.5 using HCl. As is, AMP is alkaline (industrial-grade: pKa = 9.74). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3309 ln(Concentration in % w/w) + 11.548], pH of industrial-grade AMP solutions at 1.83 % AMP w/w would be 11.7. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is, the study's maternal LOAEL could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines do NOT require any neutralization or pH adjustment, this study represents artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity.

2) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

tested as AMP ULTRA PC 2000 = high-purity grade

Species:

rat

Strain:

other: CRL:CD(SD)

Details on test animals or test system and environmental conditions:

Time-mated female rats were obtained from a commercial supplier. Sexually mature adult rats were 10-11 weeks of age and weighed approximately 200-250 grams at study start.Each animal was evaluated by the laboratory veterinarian to determine the general health status and acceptability for study purposes upon arrival at the laboratory. Rats were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle) and acclimated for at least three days. Prior to dosing animals were acclimated to the wrapping material for approximately 2, 4, and 6 hours on GD 3, 4, and 5, respectively.Animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate environmental conditions for the species. Animals were provided feed and municipal water ad libitum. Rats were stratified by GD 0 body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform mean group weights and standard deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Route of administration:

dermal

Vehicle:

other: deionized water

Details on exposure:

Rats were administered AMP once daily by the dermal (occluded) route at dose levels of 0, 30, 100, or 300 mg/kg/day for approximately 6 hours from GD 6-20. Dose solutions were prepared in deionized water at concentrations of 30, 100, and 300 mg/ml and pH adjusted to approximately 9.5 with HCl. The solutions were administered at a dose volume of 1 ml/kg body weight in order to achieve the targeted dose levels. Dose volumes were adjusted daily based on individual body weights.
Test material was utilized at a pH of approximately 9.5, authors justified as "reflecting the highest pH of most personal care and industrial formulations containing AMP". It should be noted (note from registrant) that ultra-pure undiluted AMP has a much higher pH of 12.2 at 22°C. Since the test item was partially neutralized to prepare dose solutions, the significant parental toxicity related to alcaline properties of AMP is not reflected in this study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose solutions were prepared weekly during the course of the study. Homogeneity and stability were determined prior to study initiation, and concentration of the dose suspensions was determined from the first mix.

Details on mating procedure:

Sexually mature, adult virgin females were naturally mated with males of the same strain at the supplier's facility. Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier, and maintained in the study record. Rats arrived in the testing laboratory on GD 1 or 2.

Duration of treatment / exposure:

GD6-20

Frequency of treatment:

6 hours/day

Duration of test:

15 days

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26 time-mated female rats/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The maternal and developmental toxicity potential of 2-amino-2-methylpropanol (AMP) was evaluated. Groups of 26 time-mated female CD rats were administered AMP in deionized water (pH 9.5) by the dermal route at targeted dose levels of 0, 30, 100, or 300 mg/kg/day from gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain, and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external alterations. Approximately one half of the fetuses were examined for visceral alterations while skeletal examinations were conducted on the remaining fetuses. Blood samples were collected on GD 20 from four presumed pregnant females per group to evaluate blood levels of AMP and confirm its dermal absorption.

Maternal examinations:

A cage-side examination was conducted twice daily, at approximately the same time each day to detect significant clinical abnormalities that were clearly visible upon a limited examination, and used to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Following removal of the wrapping material at the end of each daily six-hour exposure period, the test sites were graded for erythema, edema, scaling, and fissuring. Body weights were recorded on GD 0 by the supplier, and daily during the dosing period, and at necropsy (GD 21). Feed consumption were measured and statistically analyzed for the following intervals: GD 3-6, 6-9, 9-12, 12-15, 15-18, and 18-21. On GD 21, all surviving females (not fasted) were euthanized by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies was counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification. The maternal necropsy included an examination of the external tissues and all orifices. The stomach, liver, and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of tissues was not conducted. Toxicokinetic SubgroupThe first four presumed pregnant females from each dose group were selected for blood collection to evaluate systemic exposure following dermal administration of AMP. On the last day of dosing (GD20), following approximately three hours of dermal exposure, the designated animals were anesthetized using isoflurane and blood was collected from the orbital sinus. Approximately 0.5 ml of blood from each animal was collected into heparinized tubes. The samples were submitted to the analytical chemistry department for analysis and were stored frozen at approximately -80 C. AMP present in the blood was derivatized with pentafluorobenzoyl chloride under basic conditions and extracted in toluene. Quantitation was performed utilizing an isotopically labeled internal standard (D6-AMP) and matrix standards prepared in control blood. The limit of quantitation was 15 ng/g blood AMP.

Ovaries and uterine content:

A detailed examination of the reproductive tract was performed and the number and position of implantations, viable fetuses, dead fetuses, and resorptions were recorded. Resorptions were classified as either "early" or "late" based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a "dead fetus" would indicate a very recent death as evidenced by a lack external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea were counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Blood sampling:

Toxicokinetic Subgroup: Blood samples were obtained from four presumed pregnant females on the last day of dosing following approximately three hours of dermal application. AMP blood concentration was determined.

Fetal examinations:

The sex of all fetuses was determined and the body weight of all viable fetuses recorded. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum, and tail. At least one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder, and reproductive organs. The heads of these fetuses were removed, placed in Bouin's fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue. The remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone. After staining, skeletons were macerated and cleared. A thorough evaluation of the fetal skeleton was conducted on the remaining fetuses not selected for visceral examination. All fetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The fetal examinations were conducted such that investigators were blind to treatment group assignment.

Statistics:

see below

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Dermal local effects occurred at 300 mg/kg/day: a large majority of animals had slight to severe scaling and fissuring of skin site (slight: 24/26; moderate to severe: 9/26), and scabs (20/26 animals).
Scaling and fissuring also appeared a lower doses, but slight only, and transient, and was considered non adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Toxicokinetic Subgroup
In blood samples taken on the last day following approximately three hours of dermal application, results indicate dermal absorption of AMP in a dose-responsive manner. The disproportionately higher mean blood concentration of AMP at 300 mg/kg/day may have been the result of a compromised skin barrier, as evidenced by significant dermal effects (see above).
Mean AMP concentration in blood was 31, 68 and 732 ng/g at 30, 100 and 300 mg/kg/day, resp, with very high individual variability at the high-dose and a correlation with skin integrity.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

dermal irritation

Remarks on result:

other: local NOAEL

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

dermal irritation

Remarks on result:

other: local LOAEL

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

systemic NOAEL

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a rat dermal developmental toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP had no effect on development up to 300 mg/kg bw/day.

Executive summary:

In an OECD 414 study, pregnant CD rats were topically administered AMP (adjusted to pH 9.5) at dose levels of 0, 30, 100 or 300 mg/kg bw/day for 6 hours/day over GD 6-20.

• 300 mg/kg bw/day produced local effects: a large majority of animals had slight to severe scaling and fissuring of skin sites (slight: 24/26; moderate to severe: 9/26) and scabs (20/26 animals). There was no maternal systemic or developmental toxicity at any dose level tested.


• 30 and 100 mg/kg bw/day also produced scaling and fissuring, but slight and transient, which was thus considered non adverse.

Under the conditions of this study, the NOAEL for maternal local toxicity was 100 mg/kg bw/day. The NOELs for maternal systemic toxicity and developmental toxicity were both 300 mg/kg bw/day, the highest dose level tested.
Analyses of blood samples confirmed systemic exposure to AMP in a dose-responsive manner and largely supra-proportional systemic exposure at the top-dose due to altered skin barrier. The study was however not designed to quantify percent dermal absorption.

Below conclusions were drawn post-report by the registrant:

1) At the local maternal LOAEL of 300 mg AMP/kg bw/day, AMP concentration in topical solution was 342 mg/mL (see report p. 31) i.e. 34.2%, and solutions were adjusted to pH 9.5. As is, AMP is alkaline (high-purity-grade: pKa = 9.70). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3189 ln(Concentration in % w/w) + 11.401], pH of high-purity-grade AMP solutions at 34.2% AMP w/w would be 12.5. See IUCLID § 4.20: maximum human skin pH is 7 to 8 (Shu-Hua K, 2020) and substances with pH ≥ 11.5 should be considered as Skin Corrosive category 1 and not tested by dermal route (REACH, 2006 amended + ECHA, 2017). Thus, if AMP solutions hadn't been pH-adjusted, the study's maternal LOAEL could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines do NOT require any neutralization or pH adjustment, this study represents artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity.

2) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Species:

rabbit

Quality of whole database:

very high

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

very high

Additional information

Influence of pH adjustment on relevance of toxicology studies (see pH and pKa, IUCLID § 4.20/4.21):

Bibliography on maximum tolerated pH, together with pH/concentration curves for solutions of industrial and high-purity AMP grades (Fernandes, 2023), allow to conclude that repeated administration of non-neutralized AMP solutions above:

• 0.0005% w/w for oral route (reaching pH 9, maximum tolerated pH by oral route according to Turner, 2011), or


• 0.8% w/w for topical application (reaching pH 11.5, maximum tolerated pH for skin according to Shu-Hua 2020, REACH regulation 2006, ECHA 2017),

would lead to animal death or severe dose-limiting suffering. In almost all AMP in vivo toxicology studies (see IUCLID § 7.5.1, 7.5.3, 7.8), neutralization of AMP (pH adjustment or testing as AMP-HCl) allowed to test much higher concentrations and (in some studies) observe toxic effects. Since OECD guidelines do NOT require any neutralization or pH adjustment, such studies represent artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity.

This is confirmed experimentally by the 4 in vivo repeat-dose studies (see IUCLID §7.5.1 and 7.5.2) where AMP was tested without pH adjustment (there is no developmental toxicity study without pH adjustment):

• A 13-week oral rat study (Pittz 1977/79) was done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralization to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralizing AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.


• In a 5-day study on rats inhaling non-neutralized AMP aerosols (Sullivan 2017), local corrosion to skin and respiratory tract occurred at lower airborne concentrations (LOAEC: 700 mg/m3) than liver toxicity (NOEC: 700 mg/m3, LOEC: 1400 mg/m3, minimal grade liver vacuolation). pH of the 10% aerosol was calculated as 12.3 using the pH curves. Thus, protecting against local effects upon inhalation will protect against systemic effects.


• In 5-day oral studies in rats and monkeys (Pittz 1977) where non-neutralized AMP reached pH >11, mortality occurred in both species. The maximum tolerated dose (MTD) in mg AMP/kg bw/day were: well below 500 in female monkeys; 500 in female rats; 1000 in male rats. No NOAEL could be set in either species due to a 10-day recovery before necropsy and liver examinations.

 

Ingestion (only route with reproductive effects and adverse liver toxicity) is a negligible exposure route for REACH identified uses:

Reproductive effects were only observed after direct ingestion of AMP (see §7.8.1, 7.8.3), and not in two dermal studies (see §7.8.2). In humans, direct ingestion only happens via the OTC drug pamabrom and such US pharma uses are out of scope of REACH/GHS/CLP. AMP is not an approved food additive in EU and is not used to treat drinking water so REACH identified uses do not involve direct ingestion of AMP. AMP is extremely water soluble, readily biodegradable and non-bioaccumulative so no significant oral exposure to AMP via environmental emissions is expected from REACH identified uses. For all these reasons, toxicology data acquired by oral route are an extreme, barely realistic worst-case of actual systemic toxicity potential by exposure routes related to REACH uses. Indeed, by skin contact and inhalation, systemic exposure potential to AMP is very limited due to:

• risk management measures protecting against local corrosive effects of AMP and its industrial formulations (see IUCLID § 4.20/4.21, pH/pKa and also CSR);


• negligible volatility (see IUCLID § 4.6 Vapour pressure: 45 Pa at 20°C);


• few identified uses with a potential to generate aerosols (see IUCLID § 3.5 Use information);


• low concentration in end products (use as pH modifier, see CSR);


• only 7-17% dermal absorption through human skin (see IUCLID § 7.1.2 Dermal absorption).

Mode of Action Analysis / Human Relevance Framework

Mechanistic studies (see Toxicity to reproduction: Other studies /IUCLID § 7.8.3):

Numerous mechanistic studies have been performed between 1970 and 2020 which allow to better understand the reproductive effects of AMP-HCl or AMP.

The reproductive screening study (Carney 2005) on AMP-HCl identified that exposure for a period prior to implantation can lead to reduced litter size or complete litter loss. The pathology of the uteri in affected animals indicated that the implantation loss was occurring early on in the pregnancy. Post implantation loss is generally considered to be a developmental effect, however since this effect was not identified in the standard developmental toxicity studies additional studies were performed to characterize the effect and assess its relevance to humans.

 

Assessing dose response and time-window of susceptibility

Considering the data from the OECD 421 study, the dose response for the post implantation loss appears to be very steep: 9.7%, 6.7%, 69.7% and 100% at dose-levels of 0, 100, 300 and 1000 mg AMP-HCl/kg bw/day. Between low- and mid-doses, tripling of the AMP-HCl dose suddenly triggers 7-fold higher implantation loss than in controls. The steepness of the dose response suggests that exposure to AMP-HCl triggers a specific threshold event that is responsible for the effect leading to the implantation loss rather than a generalized systemic embryotoxicity. It is also clear that extending the dosing period prior to gestation (see also OECD 443) does not produce implantation loss at lower dose levels. Therefore there appears to be a “window of susceptibility” during which the triggering event must occur.

In order to verify if such a window of susceptibility existed, studies were conducted to determine exactly when the embryo resorption was occurring and also to identify whether there was a critical exposure window necessary for AMP-HCl to induce embryo loss (Rasoulpour 2009a/Exp1, Rasoulpour 2009b). These studies also assessed differences between dietary and gavage routes of administration to determine whether the toxicity was greater following a bolus dose compared to diet. In mating and pregnant female rats, ingestion of 300 mg AMP-HCl /kg bw/day (containing 213 mg AMP/kg bw/day):
- from PB -14 to GD 8 induced 68.5/75.9% (two studies) and 44.4% post-implantation loss by the dietary or gavage routes, respectively;
- from GD 1 to 8 by gavage divided by 3 the % post-implantation loss (14.2%);
- peri- (from GD 6 to 8) or post- (from GD 9 to 11) implantation did not cause post-implantation loss.

These results evidence that post-implantation loss is not triggered by AMP-HCl exposure during/after implantation (AMP-HCl is not directly embryotoxic), but by repeated AMP-HCl exposure before implantation. This is suggestive of a maternally-mediated mode of action, where AMP-HCl exposure for a sufficient period prior to implantation leads to physiological changes that reduce the capacity of the dam to support the implanting embryo. This may involve depletion of a factor necessary to support post-implantation embryo development. Other studies have suggested that this factor is choline and associated metabolites (phospholipids).

In this study that examined if the timing of the dose was critical, it was also noted that dosing via gavage produced a less severe degree of postimplantation loss compared to the same dose and duration via the diet (44.4% postimplantation loss compared to 68.5%). The lower level of postimplantation loss following gavage dosing is unlikely to be just a random variation since in the mechanistic studies conducted to date, oral dosing of 300 mg/kg bw AMP-HCl via the diet has consistently resulted in a mean of 70% postimplantation loss. This reduced severity following gavage is unusual since it is more often the case that the bolus dose provided by gavage administration is more effective than dosing via the diet. The fact that the AMP-HCl data run counter to this trend indicates that C_max may not be an important factor in the observed postimplantation loss, or that interactions in the gut when dosed via the diet play some role in the effect. For instance, AMP will exist in the small intestine in an ionized form (due to the pH in the intestine) therefore it is likely to be using some form of active absorption process since systemic bioavailability following an oral dose was approximately 100% (Saghir 2007). Due to the similarity between choline, ethanolamine and AMP, it is probable that the active transport process for choline uptake in the gut is being used by AMP. This uses a sodium ion dependent carrier mechanism (Hegazy and Schwenk, 1984; Huerga and Popper, 1952) and appears to be saturable. Therefore when a bolus does of AMP is administered it is possible that the active transport mechanism becomes saturated and less AMP is absorbed.

Alternatively, it is possible that involvement in metabolic processes may also be responsible for the difference in effect following dietary exposure or gavage. Toxicokinetic studies of AMP in rats (Saghir 2007) have demonstrated that the majority (ca. 70%) of a single oral bolus dose is excreted in the urine, un-metabolized, within 24-48 hours. The excretion of the remainder of the dose (un-metabolized) takes up to 140 additional hours. This long beta-phase elimination kinetics is consistent with the knowledge that AMP may become incorporated into phospholipids within the liver, delaying excretion. Since the synthesis of phospholipids will be increased immediately following food intake, it is possible that dosing via the diet results in a greater incorporation of the AMP into phospholipids compared to dosing via gavage, which is done in the morning, several hours after feeding takes place. Dosing via the diet could also lead to additional interactions between AMP and dietary components such as choline or ethanolamine in the gut, reducing their uptake. Therefore a greater proportion of the dose would become incorporated into endogenous processes following dietary consumption compared to gavage. This indicates that non-dietary dosing is likely to be less effective at producing toxicity and this is important when considering the relevance of these effects to the non-oral exposure paradigm in humans.

 

AMP-HCl is not directly embryotoxic

To understand if AMP-HCl was capable of directly inhibiting embryo growth, two whole embryo culture assays (Rasoulpour 2008; Rasoulpour 2010 Tier I) were conducted. These assays showed that embryos cultured in serum containing AMP (at concentrations up to three-fold higher than measured in rats given 300 mg/kg/day AMP-HCl; 3, 9, or 27 μg/mL of AMP-HCl) or the serum of rats exposed to 300 mg/kg/day AMP-HCl for at least three weeks showed no signs of embryotoxicity and developed normally. These data indicate that under the conditions of these studies, AMP-HCl was not directly toxic to the developing embryo, nor did AMP-HCl appear to affect the ability of maternal serum to support an embryo. This is consistent with the absence of developmental effects in surviving offspring in the reproductive toxicity screening study and the two-generation study. Since AMP-HCl is not directly toxic to the developing embryo then it can be concluded a maternally mediated effect is most likely driving the implantation loss.

 

Interactions with Choline and Postimplantation loss at levels that induce liver toxicity

Due to the structural similarity of AMP to the essential nutrient choline, it was hypothesized that AMP may be interacting with choline metabolism or uptake, perhaps leading to a depletion in maternal choline levels. In an in vitro study, AMP-HCl was found to inhibit the uptake of choline into CHO cells supporting this hypothesis (Stott  2006b). A NOEC could be set in this study (0.8 mM = 100 mg/L AMP-HCl containing 71 mg/L AMP). McKim (2020) confirmed that AMP alone also inhibits uptake of choline into CHO cells, and in this study AMP showed a plateauing of effects at >=0.3 mM. Note: inhibition of choline uptake is not an adverse effect, but a mechanistic effect.

Choline deficiency has been linked to fatty liver and increases in postimplantation loss since choline is a vital component of cell membranes, lipid transport and signaling molecules. Humans are less susceptible than rodents to choline deficiency due to greater capacity to synthesize it; therefore if the effects were due to choline deficiency then humans would likely be less sensitive than rats.

Two studies were performed to assess the interaction with Choline and AMP-HCl and the significance for implantation loss. In the first study (Stott 2006a) the effect of AMP-HCl administration on the choline pools in the liver was assessed. Two groups of 6 female rats were fed either a control diet or a diet containing 300 mg/kg bw/day AMP-HCl for 2 weeks prior to breeding through to GD13, then sacrificed and examined for evidence of implantation loss. Samples of livers were either fixed for pathology or frozen and used to assess the levels of choline (Cho), betaine, glycerophosphocholine (GPCho), phosphocholine (PCho), phosphatidyl choline (PtdCho), and sphingomyelin (SM). Post implantation loss was observed (67%) in the AMP-HCl treated rats in conjunction with evidence of fatty liver (hepatocyte vacuolation) and a decrease of between 20-25% in PCho. GPCho was elevated 2-3 fold compared to control dams indicating a conversion of PtdCho to Cho. All other metabolites were at similar levels to control. These data indicated that AMP-HCl treatment was capable of altering choline homeostasis in the pregnant dams. When combined with the known choline depression caused by pregnancy, these data suggest that previous findings of hepatic lipid accumulation and post-implantation loss in the OECD 421 study (Carney 2005) were secondary to phosphocholine deficiency.

 

 

Essential nutrient choline is an antidote for AMP-HCl's toxic effects

Three independent studies (2 in vivo and 1 in vitro) demonstrated that AMP-HCl effects are prevented by sufficient choline supply via the diet.

 

Rasoulpour (2009a, Experiment 2) showed that diet supplementation with choline ameliorated the degree of postimplantation loss in AMP-HCl treated rats (39% loss at 9000ppm choline vs. 66% loss at 1800ppm choline, always same AMP-HCl dose of 300 mg/kg bw/day from 2 week pre-breeding to GD 8). In rats fed 9000ppm dietary choline, a dietary choline/AMP molar ratio of 2.7 allowed a halving of post-implantation loss (once corrected for a baseline of ~10% in controls, see Carney 2005). In contrast, a 5-times lower molar ratio of 0.5 (group at 1800 ppm choline in diet) was without effect on AMP-HCl induced post-implantation loss.

 

Morin (1970) also found in vitro that a choline:AMP ratio of 50:1 completely removed AMP's inhibitory effect on endometrial phospholipids synthesis (mostly phosphatidyl choline) while a ratio of 1:1 had no protective effect.

 

Wells (1961) had already identified choline's preventive effect concerning AMP's liver toxicity. In rats fed a choline-deficient diet, AMP inhibited synthesis of lipid-bound choline from its precursors (L-methionine, formate, glycine or DL-Serine) but dietary choline supplementation, at a choline:AMP molar ratio of 0.2, completely prevented this inhibition.

Authors of these three studies independently agreed that the choline:AMP interaction is not a simple competition notably in terms of oral absorption from diet.

It is therefore clear that the choline:AMP ratio is an important factor for AMP-HCl's toxic effects. It may seem interesting to determine the choline:AMP molar ratio under realistic dietary exposure conditions. A qualitative conclusion is that this ratio is naturally considerably higher than 2.7. Indeed, dietary exposure to AMP is unrealistic based on its identified uses and intrinsic properties (see discussion on this topic under "Additional information" above), while choline is a well-known essential nutrient in rats and humans.

 

Indeed, regular and sufficient choline supply is needed for proper liver function and to avoid nonalcoholic fatty liver disease (NAFLD), a disease which is similar to AMP’s toxic effect in liver (NIH 2022. Choline - Fact Sheet for Consumers). Recommended dietary supplies notably include: 400-425 mg/person for women from 14 years, 450 mg for pregnant women, 550 mg for breastfeeding women.

 

Relationship between hepatotoxicity and implantation loss

In the OECD 421 study, AMP-HCl treated male and female rats had increased accumulation of lipid vacuoles in periportal hepatocytes at doses lower than those leading to postimplantation loss. There are a number of publications that have examined this effect of AMP on hepatocytes (Hume 1965; Russell 1965; Yue 1970; Wells 1961; Akesson 1977) and it is hypothesized to be due to disruption of lipid transport out of hepatocytes subsequent to interference with phospholipid production, inhibition of ethanolamine and choline uptake into hepatocytes and inhibition of de-novo choline synthesis. The transport of lipids out of hepatocytes is dependent on the production of the phospholipids that package them, such as phosphatidyl choline and phosphatidyl ethanolamine. Interfering with the production of these subsequently inhibits the release of lipids from hepatocytes. Interference in phospholipid synthesis has been reported in other tissues such as swine coronary arteries in vitro (Morin 1969), rabbit and human endometrial tissues in vitro (Morin 1970), house fly larvae (Bridges and Ricketts, 1967), and murine fibroblastsin vitro (Schroeder 1980). The significance of these findings towards the reproductive toxicity of AMP is unclear since the studies used high doses of AMPin vitrothat would be very difficult to achieve systemically in vivo particularly considering the irritancy of the AMP base. This effect of AMP-HCl is the only other consistent effect observedin vitroand with repeated oral dosing of AMP-HCl to animals so it is plausible that it is related to the increase in postimplantation loss. This is supported to some degree by the research done into the importance of phospholipids in embryo implantation, particularly in relation to their spatial and temporal distribution during the implantation phase and their function as a source of fatty acids such as arachadonic acid (Burnum et al.,2009).

Whilst the available data do not allow a clear and direct association between the hepatotoxicity and reproductive toxicity to be made they do indicate a potential non-specific, physiological disturbance may be occurring in the tissues, and that the reproductive toxicity could be secondary to this. The available data also indicate that the liver is more sensitive to this effect than other tissues since effects on the liver were observed at doses lower than those producing effects on post-implantation.

 

AMP-HCl has a maternally mediated mode of action

In a final study (Rasoulpour 2010), animals were treated with AMP-HCl at 300 mg/kg/day for two-weeks pre-breeding, through breeding and up to GD 6 (i.e., at the start of implantation). Extensive histopathology of the implantation sites and gene array data of the decidual swellings were generated. The purpose of this study was to examine the maternal/embryo interface at implantation in greater detail to identify whether effects in the uterus could be causing the implantation loss.

The histopathology of the implantation sites indicated the presence of vacuoles in the uterine cells immediately adjacent to the embryo in AMP-HCl treated dams, but not in controls. Staining with Oil Red O confirmed that these vacuoles did not contain lipids thus the nature of these vacuoles is unknown; however, since they appeared only in treated dams, they do indicate that AMP-HCl was producing a physiological effect within the uterine tissue. The severity of vacuolation varied across the uterus and the dams, with some implantation sites more heavily vacuolated than others. One issue with interpreting this finding is the lack of published data on what actually happens during an embryo resorption. For instance, this vacuolation could be a natural part of the resorption process, or alternatively it could be evidence of an adverse physiological effect that is causing a resorption to occur, nevertheless, the embryos in the implantation sites appeared completely normal indicating that the vacuoles precede resorption. In the repeated dose oral toxicity studies in dogs, rats and mice there have not been any reported effects observed in the uterine tissue indicative of a toxic effect. This lack of any observations in the uterine tissues in three species in the standard repeated dose protocols indicates that the uterus is not a specific target organ for toxicity, at least in the absence of pregnancy.

Gene expression arrays of the cells taken from decidual swellings from treated and control dams were analyzed to identify if there was any effect of AMP-HCl administration at the transcriptional level. The results of the analysis indicate that certain genes were less active in AMP-HCl treated rats compared to control. Specifically these genes (e.g., claudins and occludins) are associated with the formation of tight junctions. It is possible that this observation is associated with the postimplantation loss, particularly since these changes are occurring at the start of implantation rather than during or subsequent to the postimplantation loss. By influencing the formation of tight junctions, the decidual swelling surrounding the embryo could be made more ‘leaky’ and implantation sites may not be completely isolated from the maternal circulation. This, for example, could result in penetration of the maternal immune system into the implantation site leading to rejection and resorptions of the embryo.

When interpreting this data it is important to understand that this level of detailed gene array analysis has not been performed on other implantation sites where resorption of embryos is suspected. It is also not possible to state whether AMP-HCl directly or indirectly impacted gene transcription in the cells analyzed. If AMP-HCl was capable of directly interfering with the transcription of genes involved in tight junction formation it seems plausible that adverse effects in other tissues would be noticed, and this is not the case. Therefore it is not possible to concretely conclude that the lower gene activity observed is a consequence of AMP-HCl exposure or preparation for the resorption of an embryo; i.e. the changes in gene activity could be casually related rather than causally related to the observed effect. Further research would be needed to fully understand the relevance and importance of this information to the postimplantation loss observed.

This study provides additional data supporting a maternally-mediated mode-of-action for AMP-HCl-induced postimplantation loss, which may involve disruption of normal implantation through alterations in the tight junction signaling network.

 

Conclusions of mode of action experiments

Oral administration of AMP-HCl to rats prior to and during pregnancy is associated with an increase in postimplantation loss via a threshold, maternally mediated mechanism. The postimplantation loss occurs during or shortly after implantation (gestation day 6 to 8), and requires exposure to AMP-HCl for a period of at least 8 days prior to implantation via a dose route that provides significant systemic exposure (oral and only after pH neutralization using AMP-HCl). There is a clear threshold for this effect and the dose response is very steep, but very consistent (multiple AMP-HCl mechanistic studies using the same dose level have consistently produced the same degree of implantation loss). Considering the evidence of other toxicological effects at doses producing post-implantation loss (alteration in phospholipid synthesis in the liver leading to fat accumulation, gastrointestinal irritation) it is plausible that the post implantation loss could be secondary to a more general physiological effect in the dam rather than a specific embryotoxic effect. Taking this into consideration, AMP-HCl appears to indirectly affect the implanting embryo via a mechanism that involves maternal toxicity.

In a dermal developmental toxicity study there were no observed effects on any developmental parameter. The lack of postimplantation loss observed in that study is consistent with the lower systemic availability of AMP following dermal dosing, coupled with the shorter dosing period that covered gestation day 6 through to 20. However the highest dose that could be used in this study was limited by irritation to 300 mg/kg bw/day. Based on the applied dose, the maximum possible systemic dose from this study would have been approximately 100 to 150 mg/kg bw based on the absorption through the skin of between 30 to 40% AMP in the rat. Considering the significantly lower bioavailability via dermal dosing and potential need for oral exposure, even if this study had dosed for a longer period prior to gestation day 6, it is very unlikely post implantation loss would have occurred.

Thus this reproductive toxicity appears only relevant to those exposure routes where significant systemic availability is possible (oral). It is also important to note that dermal penetration data comparing human and rat skin demonstrated that penetration of AMP through human skin was less than half that observed in rat skin. As such exposure of humans dermally to AMP would be very unlikely to lead to sufficient systemic exposure to re-create the toxicity observed following oral dosing. Dermal exposure to AMP through industrial/professional/consumer uses would also be to the salt form (AMP is used as a neutralizer) and penetration of the salt through the skin will be lower than the base due to differences in lipophilicity, further reducing the potential for systemic exposure.

 

Extra literature on mechanism (not summarized in IUCLID as not involving AMP, rats or humans):

Bridges, R.G. and Ricketts, J. (1967). The incorporation, in vivo,of aminoalcohols into the phospholipids of the larvae of the Housefly,Musca domestica. J. Insect Physiol. 13,835-50

Burnum, K.E., Cornett, D.S., Puolitaival, S.M., Milne, S.B., Myers, D.S., Tranguch, S., Brown, H.A, Dey, S.K and Caprioli, R.M. (2009). Spatial and temporal alterations of phopholipids determined by mass spectrometry during mouse implantation. J. Lip. Res. 50, 2290-8

Hegazy E. and Schwenk M. (1984). Choline uptake by isolated enterocytes of guinea pig. J. Nutr. 114, 2217-20,

de la Huerga, J. and Popper, H. (1952). Factors influencing choline absorption in the intestinal tract. J. Clin. Invest. 31(6), 598–603.

Hume, J.W., Mulford, D.J. and Russell, P.J. (1965). Effect of 3-aminopropanol on choline deficiency in rats. Proc. Soc. Exp. Biol. Med. 118(2), 430-3

Morin, R.J. (1969).In Vitro inhibition by metabolic antagonists of Phosphate-³²P into the major phospholipids of swine coronary and pulmonary arteries. Atheroscler. Res. 10(3), 283-9

Schroeder, F. (1980). Regulation of aminophospholipid asymmetry in murine fibroplast plasma membranes by choline and ethanolamine analogues.Biochim. Biophys. Acta. 599(1), 254-70

Justification for classification or non-classification

Post-implantation loss was the only adverse reproductive toxic effect noted in a larger-than-required dataset of fertility, developmental and mechanistic studies.

It was observed exclusively in rat studies on neutralized form AMP-HCl at 213 mg AMP/kg bw/day (2180-2620 ppm AMP in diet, Carney 2005 and multiple AMP-HCl mechanistic studies at same dose). It does not warrant Reproductive Toxicity classification for AMP, from regulatory and scientific points of view:

1) Effects don't meet REACH/CLP regulatory criteria:

• Post-implantation loss only occurred with AMP-HCl (CAS N° 3207-12-3) and not AMP (CAS N° 124-68-5), so use for classification of AMP needs to consider the influence of the hydrochloride salt which neutralizes AMP (alkaline test item);


• 2180-2620 ppm AMP cannot be reached without AMP neutralization due to intolerable pH (calculated as 10.9-11.0 using an experimental pH curve, Fernandes 2023);


• As test item neutralization is not required in OECD guidelines referenced by REACH regulation, all studies on AMP-HCl represent artificial dose maximization in excess of REACH principles, removing AMP's critical toxic effect which is pH-dependent non-specific toxicity;


• This is confirmed by 13-week oral rat studies done with and without AMP neutralization (Pittz 1977/79 x2, see UCLID § 7.5.1), where neutralization artificially increased AMP’s maximum tolerated dose by a factor of at least 3.5;


• Where investigated in oral studies, parental/maternal liver toxicity was systematically present (OECD 443, 421, OECD 414 rabbit and rat, multiple mechanistic studies); in OECD 421-type studies, it was present at 3-fold lower dose-levels than post-implantation loss. Multiple mechanistic studies (see 7.5.1 and 7.8.3) suggest these liver and reproductive effects share a common pathway: they are both associated with or correlated to, perturbation of choline uptake and choline metabolism into phospholipids. The effect is clearly maternally mediated and AMP is not directly toxic to the embryo or developing fetus after implantation. All surviving embryos develop normally through to sexual maturation and subsequent mating.


• According to GHS/CLP criteria, reproductive effects secondary to general toxic effects do not warrant Reproductive toxicity classification. 

2) There is no relevant hazard to human:

• Post-implantation loss was not observed in the OECD 443 study, which includes >2x more animals per dose, 2-3x longer treatment duration compared to OECD 421, and 2 generations vs. one;


• No reproductive effects were reported in 6 human clinical trials on pamabrom (containing 25.6% w/w AMP), two of which were on pregnant women and three of which were on women with periods;


• No adverse effects on reproduction are mentioned for any of the 20 OTC drugs containing pamabrom. The only mention related to reproduction is a generic statement used for almost all drugs: "If pregnant or breast-feeding, ask a health professional before use."


• Pamabrom drugs are used since 7 decades, establishing strong pharmacovigilance.

3) There is no relevant risk to human:

• A clear and high NOAEL for post-implantation loss could be identified when considering OECD 443 and 421 studies combined (142 mg AMP/kg bw/day) and the specific susceptibility period (time-window) for post-implantation was also identified in mechanistic studies;


• The dose-response for post-implantation loss is very steep, in OECD 421 study tripling of the dose elicited a 7-fold increase in post-implantation loss (all-or-nothing); therefore the NOAEL is very reliable and the safety margin for humans is both reliable and large;


• As reproductive effects occur at higher dose-levels than liver toxicity, protecting against liver toxicity (via risk assessment using the long-term systemic oral DNEL which is based on liver effects) will automatically protect against any reproductive effects;


• The potential for exposure of reproductive organs to AMP is negligible under realistic exposure conditions (skin contact and inhalation) based on local corrosion requiring strong risk management measures, negligible volatility, few uses generating aerosols, low concentration in end products, and only 7-17% dermal absorption through human skin;


• The mechanism of AMP's liver toxicity and AMP-HCl's reproductive toxicity has been thoroughly investigated and expresses only when AMP(-HCl) is in large excess of choline. Depending on the choline:AMP ratio, choline can prevent AMP’s inhibition of liver choline synthesis (Wells 1961, dietary choline), prevent AMP's inhibition of endometrial phospholipids synthesis (Morin 1970, in vitro) and halve the magnitude of AMP-HCl’s effect on post implantation loss (Rasoulpour 2009a, Experiment 2, dietary choline). Therefore, AMP’s toxicity is largely indirect and has an identified antidote: choline;


• Choline is an essential nutrient so it will counter-act the toxicity of any trace oral exposure to AMP. Indeed, regular and sufficient choline supply is needed for proper liver function and to avoid nonalcoholic fatty liver disease (NAFLD), a disease which is similar to AMP’s toxic effect (NIH 2022. Choline - Fact Sheet for Consumers. https://ods.od.nih.gov/factsheets/Choline-Consumer/). Recommended dietary supplies notably include: 400-425 mg/person for women from 14 years, 450 mg for pregnant women, 550 mg for breastfeeding women;


• Ingestion is the only route with observed post-implantation loss and is a negligible exposure route to AMP for REACH identified uses, be it direct or indirect (via the environment); therefore, the choline:AMP ratio from human oral exposure will be largely above the ratios identified to provide full or partial prevention of AMP(-HCl) adverse effects;


• Although AMP cannot be used in food contact in EU, absence of risks upon low-dose AMP ingestion has been concluded by renowned non-EU regulatory bodies: US FDA approved AMP in various food contact applications (adhesives, coatings and fillers in paper/paperboard, certain resinous and polymeric coatings) and AMP is in China’s GB 9685 positive list (additives for food contact materials).