Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WEDO Shangai, batch No. 24-10-2017
- Expiration date of the lot/batch: 24/10/2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility and stability of the test substance in the solvent/vehicle: 6 hours at room temperature with the concentration of 1 mg/mL and 50 mg/mL in corn oil

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulation were prepared daily before dose adminitration.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standatd laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Age at study initiation: 9 weeks
- Weight at study initiation: Males: 246.08 - 290.96 g; Females: 200.29 - 240.94 g
- Fasting period before study:
- Housing: Animals were housed in a standard polypropylene cage with stainless steel mesh top grill having facilities for holding pellet food and drink water bottle fitted with stainless steel supper tube. Clean sterilized paddy husk was provided as bedding material. During acclimatiozation, two animals of the same sex were housed.
i. Pre mating: Two animals of the same sex and group per cage were housed
ii. Mating: During mating, two animals (one male and one female) of the same group per cage were housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: All the animals were identified within the cage by tail marking using a permanent marker pen. Additionally, a cage card was displayed which included study no., cage no., sex, animal no. start date and end date of acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature (19.1 to 22.9°C)
- Humidity (%): 44 - 68
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Required guantity of test item (Table 1) was weighed into a clean glass beaker and there by addinf little volume of vehicle into a beaker, mixed well with clean galss rod and transferred to a measuring cylinder. The volume was made up to the required quantity with the vehicle to ger desired concentrations of 12.5, 25 and 50 mg/mL of test item for low, mid and high dose groups respectively. The quantity of the test item taken and the volume prepared varied depending on the requirement.

VEHICLE
- Justification for use and choice of vehicle: The test item was not clearly miscible with distilled water and clearly miscible with corn oil at concentration of 50mg/mL as evidence by the in-house miscible test results. Hence, corn oil was selected as a vehicle for test item formulation preparation. Corn oil is universally accepted vehicle used in pre-clinical toxicity studies.
- Concentration in vehicle: 1 mg/mL and 50 mg/mL
- Amount of vehicle (if gavage): 5 mg/mL
- Lot/batch no.: A1903001
Details on mating procedure:
- M/F ratio per cage: 1:1
- Lenght of cohabitation: average 14 days
- Proof of pregnancy:sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): After confirming presence of sperm in tha vaginal smear (Day 0 of pregnancy), tha mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose samples of 3-(isodecyloxy) propylammonium acetate were analzed for dose concntration verification by validated GC method. The results are found to be within the acceptable range of ± 15% to the nominal concentation with RSD less than 10%.
Duration of treatment / exposure:
The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post mating period (total of 39 days of treatment)
The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).

The test item/vehicle were administered by oral route using stainless steel intubation cannula attached to a disposable sytinge. As the test substance is classified as corrosive 1B, all the test doses were admnistered in a equivolume of 5mLúkg body weight with the concentration of 12.5, 25 and 50 mg/mL for low, mid and high dose groups, respectively to minimize the concentration of the test item formulations. Vehicle was administered to vehicle control group at an equivolume of 5 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation.
Frequency of treatment:
The test item item formulation or vehicle were administered to animals through oral (gavage) route once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males + 12 females

Examinations

Parental animals: Observations and examinations:
CLINICAL SIGNS OF TOXICITY AND MORTALITY/MORBIDITY
- All animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity. Dead / animals were necorpsied at the earliest.

BODY WEIGHT
- The animals were weighed at receipt , on the first day of dosing, weekly thereafter and at termination.
- The females were weighed on gestation days 0, 7, 14, and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period and on day 14 (fasting body weight).

FEED CONSUMPTION
- Feed consumption was measured for all animals once a week during premating and once for males during post matinf period. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded durinf gestation days 0 to 7, 7 to 14 and 14 to 20 and on location days 1 to 4, 4 to 7 and 7 to 13.

CLINICAL PATHOLOGY - HORMONE ANALYSIS: BLOOD COLLECTION FOR TOTAL T4 (THYROXINE) LEVEL ESTIMATION
- Blood samples were collected from all animals for measurements of serum total T4 levels on the following shedule:
a) two pups per liiter on lactation day 4
b) All dam at termination (lactation day 14)
c) Two pups (same sex per litter) at termination (lactation day 14)
d) All adult males, at termination (after completion of 39 days of treatment)
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored for two weeks after five days of acclimatization to evaluate its oestrus eyclicity (4 to 5 days). Only females with norma) oestrus cyclicity were selected for the treatment. Vagina! smears were monitored daily from the beginning of the treatment period until evidence of mating for all surviving females. When obtaining vaginal/cervical eells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females.
Sperm parameters (parental animals):
No data available
Litter observations:
- The duration of gestation was recorded and calculated from day 0 of pregnancy. The day of littering was considered as lactation day 1.
- The number of pups born (dead and live) in a litter, sex, live births and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 were recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4) and the ratio of AGD to the cube root of pup body weight was calculated. All survived male pups were examined for appearance of nipples/areolae on postnatal day 13 (lactation day 13).
- The litter was observed daily in order to note the number of alive, dead and cannibalized pups. Ali the dead and sacrificed pups were examined and subjected to gross pathological examination.
- Fertility index for dams, sires and pup live birth index, mean litter size per group, survival index and sex ratio at birth were calculated.
Postmortem examinations (parental animals):
SACRIFICE
- The male were sacrificed after completion of 39 days of treament, females were sacrificed on lactation day 14.
- All surviving animals were fasted overnight, water was provided ad libitum during fasting.


GROSS NECROPSY
- The vaginal smear of females on the day of necroscopsy (lactation day 14) was performed and stage of oestrus cycle was recorded.
- The animal were euthanized using deep CO2 asphyxiation and subjected to gross necroscopsy, external and internal pathological examination.


HISTOPATHOLOGY / ORGAN WEIGHTS
- Detailed histopatological examination was performed on the ovaries, testes and Epididymis of the animals from th control and the high dose group including macroscopically abnormal tissues of all animals, whether died during the study or sacrificed at termination.
- The histopathological examination was not extended to the lower dose groups as there were no treatment-related effects observed at the high dosc level (target organs).
- The other preserved organs including thyroid was not examined as there were no treatment related changes observed in high dose group. These tissues were ernbedded in paraffin wax, sectioned at 4 to 6 micrometers and stained with hematoxylin and eosin. The testes were sectioned at 3 to 4 micrometers and stained with hematoxylin and eosin sta in and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS sta in aided spermatogenesis evaluation .
Postmortem examinations (offspring):
SACRIFICE
- The pups were sacrificed on lactation day 13.
- These animals (sacrificed and dead) were subjected to postmortem examinations as follows:
a) PND 4 (for blood collection)
b) PND 13


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including:
a) Thyroid along with parathyroid
b) Gross lesions
Statistics:
The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was vcrified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests designated by the superscripts throughout the report as stated below:
* Statistically significant (P < 0.05) change than the vehicle control group.
NOTE: Data of non-pregnant females was excluded from statistical analysis.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In group G4 [250 mg/kg body weight], there were no clinical signs of toxicity noted in any of the animals of either sex for the first two days of the treatment period. However, treatment related clinical signs of toxicity like, slight to severe salivation (11 males and 6 females), soft stools (2 males), tremors (4 males and 2 females), porphyrin staining /chromoclacrorrhea (9 males and 4 females), slight to moderate wet perineum (9 males and 7 females) and hair thinning (1 male) were noted during the treatment period. Among these, 4 males (one on day 5, one on day 6 and two on day 7) and 2 females (one on day 6 and another on day 8) were found dead during pre-mating period and the remaining animals were found recovered later in the treatment period.
These noted clinical signs of toxicity at group G4 [250 mg/kg body weight] in either sex are considered as treatment related, due to noted reduced mean body weight, mean body weight gain and mean feed consumption during the treatment period at this dose level.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In group G3 [1 25 mg/kg body weight], the males did not reveal any significant reduction in mean body weight and pcrcent change in body weight. However, the females from this dose group revealed reduction in mean body weight and percent change in mean body weight during pre-mating period which is statistically significant during day 1 to 7 and 1 to 14 [percent change in mean body weight].
ln group G4 [250 mg/kg body weight], treatment related reduction in mean body weight and mean percent change in mean body weight with respect to day 1 was noted in either sex. These changes are statistically significant in males (throughout the treatment period) and statistically significant in females (during pre-mating period). However, a slight recovery was noted in both mean body weight and body weight gain at this dose level in either sex.
These noted effects in both group G3 [125 mg/kg body weight] and G4 [250 mg/kg body weight] are considered as treatment relatecl as these observed changes are
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In group G3 [125 mg/kg body weight], the mates did not reveal any significant reduction in mean feed consumption. However, the females frorn this dose group revealed a slight reduction in rnean feed consumption during pre-mating period which is not statistically significant. However, this reduction can be considered as treatrnent related due to reduced mean body weight and percent change in mean body weight during this period
In group G4 [250 mg/kg body weight], treatment related reduction in mean feed consumption was noted in either sex during pre-mating period (week 1 and 2) and these changes are statistically significant when compared with vehicle control group animals.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No treatment realted histopathological finding were noticed during microscopic eyamination conducted for epididymides or testes (in males) and for ovaries (in females) in group G4.
A detailed qualitatitve examination of rhe testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, mulnucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
Treatment related histopathological findings like, non-glandular stomach with inflammation (2 males), non-glandular stomach with necrosis (3 males), no-glandular stoamch with erosio/ulcer (1 female) and atrophic thymus (1 female) was noted durich histopathological examination in group G4.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
SERUM THYROXINE (T4) LEVELS:
There was no treatment related changes noted in serum Thyroxine (T4) hormone levels at all the tested dose group [G2 (62.5 mg/kg body weight), G3 (125 mg/kg body weight) and G4 (250 mg/kg body weight)] adult mates when compared with vehicle control group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no changes (irregularities) noted in the oestrus cyclicity of all females from group G2 (62.5 mg/kg body weight) and G3 (125 mg/kg body weight) during the entire treatment period till confirmation of mating, except one female from group G3 noted with 1 irregular cycle out of 4 cycles.
In group G4 [250 mg/kg body weight], there were no changes (irregularities) noted in the oestrus cyclicity of all surviving females during entire treatment period till confirmation of mating. However, two females which were found dead during pre­mating period, did not noted with one complete oestrus cycle.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
In group G4 [250 mg/kg body weight], the cohabitation was initiated with 8 pairs initially due to mortalities during pre-mating period [a total of 4 males and 2 females were found dead]. The other two surviving females were cohabitatecl with male at the time of availability of praven male from the same group. Due to unavailability of required number of males from this dose group, the cohabitation was continued after 14 days of initial period with thc same pair. Hence, the copu I a tory interval was effected from this dose group. A total of 3 females were confirmed with mating within 14 days of cohabitation period with the first male, 4 females were confirmed with matinf within 20 days of cohabitation period with the same first male and 3 females were confirmed with mating within 25 days of cohabitation period with the second (proven) male of the same group.

This effect in copulatory interval at group G4 (250 mg/kg body weight) is considered as treatment related due to noted clinical signs of toxicity, reduced mean body weight, reduced percent change in mean body weight and reduced mean feed consumption during pre-mating and cohabitation period in either sex.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified

Details on results (P1)

No further data available.

Effect levels (P1)

Remarks on result:
not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no changes noted in daily observation of pups at all the tested [G2 (62.5 mg/kg body weight), G3 (125 mg/kg body weight), G4 (250 mg/kg body weight)] and vehicle control [G1 (0 mg/kg body weight)] group litters during postnatal period.
However, 2 pups ( I male and l female) were found dead on PND 3 from group G4, which can be considered as incidental as there were no gross pathological changes noted in these pups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no treatment related changes observed in mean pup weight of either sex during post-natal period at all the tested dose group [G2 (62.5 mg/kg body weight), G3 (125 mg/kg body weight), G4 (250 mg/kg body weight)] litters when compared with vehicle control group.
However, statistically significant reduction in mean pup weight on PND 13 in either sex was noted at group G4 (250 mg/kg body weight) when compared with vehicle control group. This noted reduction can be considered incidental and unrelated to treatment as there were no clinical observations noted in pups of' either sex at this dose level and also no statistically significant reduction noted in mean pup weight on PND 1, 4 and 7.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no changes noted in mean ano-genitaI distance ratio of either sex measured on PND 4 at all the tested dose group [G2 (62.5 mg/kg body weight), G3 (125 mg/kg body weight), G4 (250 mg/kg body weight)] litters when compared with vehicle control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no occurrences of nipples in male pups at any of the tested dose group [G2 (62.5 mg/kg body weight), G3 (125 mg/kg body weight), G4 (250 mg/kg body weight)] litters and vehicle control group litters.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological lesions noticed during conduct of necropsy in any of the pups of either sex [dead and sacrificed on PND 4 and 13].
Histopathological findings:
not specified
Other effects:
no effects observed
Description (incidence and severity):
SERUM THYROXINE (T4) LEVELS
There was no treatment related changes noted in serum thyroxine (T4) hormone levels at all the tested dose group [G2 (62.5 mg/kg body weight), G3 ( 125 mg/kg body weight) and G4 (250 mg/kg body weight)] pups collected on PND 13 when compared with vehicle control group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

There were no changes or effects related to treatment observed in pups.

Effect levels (F1)

Key result
Dose descriptor:
dose level:
Generation:
F1
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not specified

Details on results (F2)

No further data are available

Effect levels (F2)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results, the No Observed Adverse Effect Level (NOAEL) of 3-(isopropyl) ammonium acetate was found to be 125 mg/kg body weight, the mid dose when administered to the males for two weeks pre-mating, during mating and up to the day before sacrfice during post-mating period (total of 39 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) an up to lactation day 13, under experimental conditions employed.