2-amino-2-methylpropanol

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

repeat-dose toxicity data generated in a reproductive toxicity study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 443

Principles of method if other than guideline:

repeat-dose toxicity data generated in a reproductive toxicity study. For further details see the cross-referenced study summary.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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)

Sex:

male/female

Route of administration:

oral: feed

Details on oral 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 concentration added to the pre-mix, after which the diet was blended to achieve a total batch of homogeneous diet at the appropriate concentration/group. The test diets were prepared approximately weekly, 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.

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

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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).

Clinical signs:

no effects observed

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

Description (incidence and severity):

Test substance triggered organ weight changes in kidneys and liver of F0 generation males:

KIDNEY: There was a dose-related increase in kidney weights as absolute values and percent body weight at ≥ 50 mg/kg bw/day. Differences were statistically significant at ≥ 100 mg/kg bw/day. There were no histological correlates.

LIVER: There was a dose-related increase in all three liver weight parameters at ≥ 50 mg/kg bw/day with statistical significance at 200 mg/kg bw/day. The increased liver weights correlated histologically with hepatocellular vacuolation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted, including for the number of implantation sites.

A statistically significant higher number of unaccounted for sites was noted in the 200 mg/kg/day group compared to the control group (2.7 vs. 1.0). This difference was primarily attributed to a single female in this group with 13 unaccounted-for sites. In addition, the number of unaccounted-for sites in this group was within the Charles River Ashland historical control data range, and therefore was not considered test substance-related.

F0 generation females with suspected reduced fertility (see "Reproductive performance") had quantitative counts of primordial/small growing ovarian follicles performed. Primordial/small growing ovarian follicular counts of these animals were similar to individual values obtained from the F1 Cohort 1A control females, with the exception of one animal in the 50 mg/kg bw/day group (lower ovarian follicle counts); however, this change did not occur in a dose-related manner. Test substance administration did not cause decreased primordial/small growing ovarian follicles in these F0 animals.

Neuropathological findings:

not examined

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

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

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:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

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:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

no

For further details see the cross-referenced study summary. Above summaries relate to the parental generation (F0). Below tables detail liver findings in both generations.

 

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's 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 up to 200 mg AMP-HCl/kg bw/day (containing up to 142 mg AMP/kg bw/day) over two generations. Toxicologically relevant effects were limited to liver toxicity in male rats of F0 and F1A generations: 

• 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.

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. 

Below conclusions were drawn post-report by the registrant:

1) The absence of irreversible liver lesions means effects do not warrant STOT RE classification.

2) 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.

3) 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-2-methylpropanol

EC Number:

204-709-8

EC Name:

2-amino-2-methylpropanol

Cas Number:

124-68-5

Molecular formula:

C4H11NO

IUPAC Name:

2-amino-2-methylpropan-1-ol

Constituent 2

Reference substance name:

2-Amino-2-Methylpropanol Hydrochloride

Cas Number:

3207-12-3

IUPAC Name:

2-Amino-2-Methylpropanol Hydrochloride

Test material form:

solid: particulate/powder

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.

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

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

Doses / concentrations 2

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

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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

2

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

3

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

4

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

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

2

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

3

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

4

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.