[(±)-(4-amino-2-hydroxy-4-oxobutyl)trimethylammonium] chloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 January 2013 to 30 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Age at study initiation: Young adult rats, approximately 11 weeks old at starting and 13 weeks at mating. The age range within the study was kept to the minimum.
- Weight at study initiation: Males: 366 g – 421 g, Females: 214 g - 247 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment.
- Housing: Rodents were group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Water (e.g. ad libitum): tap water from municipal supply, ad libitum.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 24.0°C
- Humidity (%): 32 - 58%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 3 January 2013 To: 18 February 2013

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in Distilled water at 15.6, 62.5 and 250 mg/mL. Formulations were prepared weekly intervals, based on the stability test performed at the Test Site. Analysis of test item formulation samples of 15.6 mg/mL, 62.5 mg/mL and 250 mg/mL showed no decrease of concentration within 15 days and can be considered as stable for at least 15 days stored in room temperature.

Details on mating procedure:

- M/F ratio per cage: Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage.
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days.
- Proof of pregnancy: A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from test item formulations and from the control on 3 occasions, during the first and last weeks and approximately midway during the treatment, one set to analyze and one set as a back-up, if required for any confirmatory analyses.

The measured concentrations of the test item evaluated for each test item-dose group varied between 97.9 and 101.5%. No test item was detected in the control samples. These results were within acceptable ranges (90 % - 110%).

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).

Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing).

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 62.5, 250 and 1000 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

12 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels and the vehicle were selected based on available data, formulation and analytical trials and information from previous experimental work, including the results of a repeated dose range finding study in the rat (the test item administered by oral gavage to Wistar rats for 7 consecutive days at dose levels of 62.5, 250 and 1000 mg/kg bw in distilled water at a dose volume of 4 mL/kg bw, was not associated with any overt adverse effects that could be clearly related to test item administration).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon (pm) as practical during the working day, as no peak period of effects was noted after dosing during the first days of treatment.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), PPD4 and PPD5 (before termination). Body weights of the female animals were additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION :
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly.

OBSERVATION OF THE DELIVERY PROCESS, OFFSPRING AND NURSING INSTINCT
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

PARAMETERS EXAMINED
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.

The pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-natal, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups.

GROSS EXAMINATION OF DEAD PUPS:
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
Gross necropsy was performed on all animals. Terminally, after completion of the treatment, animals were euthanised under pentobarbital anaesthesia, followed by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the females as applicable.

HISTOPATHOLOGY: Yes
For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups and all macroscopic findings (abnormalities) from all animals. The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution. In addition, for 5 animals/sex/group (subgroup B), the following organs and tissues, or representative samples, were preserved: Gross findings, adrenals, aorta, brain, epididymides, eyes with optic nerves, oesophagus, femur with marrow, heart, kidneys, large intestine, lacrimal glands, harderian glands, liver, lungs with bronchi, lymph nodes, mammary gland (inguinal), ovaries with oviduct, pancreas, pituitary, prostate, salivary gland (mandibular), sciatic nerve, seminal vesicles, coagulating glands, skeletal muscle (quadriceps), skin and subcutis (inguinal), small intestine, spinal cord (cervical, lumbar, and thoracic levels), spleen, sternum with marrow, stomach, testes, thymus, thyroid with parathyroids, tongue, trachea (with main stem bronchi), urinary bladder, uterus, vagina.

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

ORGAN WEIGHTS:
At the time of termination, body weight and weight of the following organs of all parental animals were determined:

- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides, prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries, pituitary

From subgroup B animals, the following organs were weighed in addition to the ones previously mentioned:

- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus.
- With a precision of 0.001 g: adrenals.

For all organs, paired organs were weighed individually. Individual and/or paired absolute organ weight are reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

Postmortem examinations (offspring):

GROSS NECROPSY
Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities. Any pups showing abnormalities in structure or behaviour, including the pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination, in order to identify the probable cause of death if possible.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Reproductive indices:

- Male mating index
- Male fertility index
- Female mating index
- Female fertility index
- Gestation index
- Pre-implantation mortality
- Intrauterine mortality

Offspring viability indices:

- Survival index of pups on postnatal Days 0 and 4

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
There was no mortality during the study. No test item-related adverse effects or systemic clinical signs were noted following administration of daily by oral gavage under the conditions of this study, or in the animals. In one female animal in the Control group thin fur on both forelimbs, left axillary, lumbar area or on the thorax ventral area was observed from Day 25. In a Low dose female thin fur on both forelimbs or on the thorax ventral area was observed from Day 30. In one female animal in the Low dose group, slight decreased activity, hunched back position, piloerection, slight opacity in both eyes and dark brownish discharge (moderate) in urogenital area was observed from Day 33.

BODY WEIGHT AND WEIGHT GAIN
No test item related effects were noted on the mean body weight and body weight gain values following daily administration of the test item at dose levels up to and including 1000 mg/kg bw/day, during the treatment period. A higher increase in body weight gain was recorded for treated males between Days 21 and 27 (p<0.05 or 0.01) compared to the control animals. However, the overall body weight gain values (Days 0-27) were comparable to the control.

FOOD CONSUMPTION
There were no test item-related differences in the mean daily food consumption in any test-item treated group (62.5, 250 or 1000 mg/kg bw/day) when compared to the Control. Compared to control, differences attaining statistical significance were noted for males at 250 mg/kg bw/day (Mid dose) and 1000 mg/kg bw/day (High dose) between Days 21 and 28 (p<0.05 and 0.01, respectively). The individual values remained within the normal ranges and the finding was not considered toxicologically significant or to reflect an adverse effect.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Microscopic evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa were similar in Control and High Dose males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no statistically significant differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with the test item administration. In all groups, the mating indices were 100%, the fertility indices 92% due to 1/12 non-pregnant females, in Groups 1, 2, 3 and 4 (these values are all in the normal historical range). The gestation indices were 100, 91, 100 and 100% values, respectively, in Groups 1, 2, 3 and 4. Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 days of pairing (cohabitation). No test item effect on the duration of pregnancy or abnormalities in the gestation outcome ascribed to the treatment were observed. The mean duration of pregnancy was similar in the control and test item treated groups, all were 20 to 23 days. All the parturitions were normal. The number of corpora lutea and implantation sites were comparable in the treated groups up to and including 1000 mg/kg bw/day with the mean value recorded in the Control group. There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 1000 mg/kg bw/day.

ORGAN WEIGHTS
There were no toxicologically significant effects on organ weights. Compared to controls, slightly lower weights of pituitary glands were recorded for males at 250 mg/kg bw/day (Mid dose). The difference was approximately 15% and attained statistical significance (p<0.05) for absolute values, body weight and brain weight related mean values. The changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as physiological variation.

GROSS PATHOLOGY
There was no evidence of the test item-related macroscopic findings at dose levels of 62.5, 250 and 1000 mg/kg bw/day. Incidental or common background observations including small testes and epidydimides, single cyst of the vagina, uterine dilation, enlargement of the spleen, raised red area of the glandular mucosa in the stomach and thin fur at the dorsal thoracic/lumbar/forelimbs regions were recorded at necropsy. All these changes were observed without notable incidence and/or cross control or treated animals and were not toxicologically meaningful.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test item-related microscopic effects were observed at dose levels of 62.5, 250 and 1000 mg/kg bw/day. Microscopic evaluation of the brains or pituitaries, male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females. Minimal to moderate tubular degeneration/atrophy and/or minimal to mild reduced sperm content in the epidydimides in 3/12 Control males, mild tubular dilatation of the left testis in 1/12 Low dose male, mild acinar cell debris in 1/12 Control male or cell mixed mononuclear infiltrate/inflammation of the prostate in 2/12 High Dose males, were regarded as incidental. All other changes were incidental or a common background.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY AND CLINICAL SIGNS (OFFSPRING)
Test item administered to parental generation at up to 1000 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities clearly ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically.

In the Control group (140 pups examined), 12 pups were found dead on PND0 from the same dam, with 6 negative floating test (possibly stillborn) and 6 were found dead and had positive pulmonary floating test; pups were not suckled. This litter was excluded from offspring evaluation. Another pup was found dead and cannibalized on PND1 without displaying any clinical signs prior to death.

In the 62.5 mg/kg bw/day Low dose group with 140 pups examined, one pup was found dead on PND0, with positive pulmonary floating test. In single pup haemorrhage on the neck, dorsal area was observed on PND0.

In the 250 mg/kg bw/day Mid dose group, with 156 pups examined, 3 pups were found dead on PND0, with negative floating test (possibly stillborn) and one pup was cannibalized (with cold, grey body clinical signs) on PND1. Haemorrhage on the neck, dorsal area (in 1 pup), on the nose (in 1 pup), on the head, cranium (in 2 pups) or on the jaw (in 1 pup); cold or grey body (in 2-2 pups) and slight scab on the nose (in 1 pup) were observed occasionally on PND0.

In the 1000 mg/kg bw/day High dose group with 151 pups examined, one pup was found dead on PND0, with negative floating test (possibly stillborn). Haemorrhage on the nose (2 pups) or on the head, cranium (1 pup) were observed on PND0 and one pup was retarded in body weight between Days 0-4.

No other external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. The incidence of these findings was low, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect. The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 when evaluated as litter data were comparable to control values at up to and including 1000 mg/kg bw/day, or showed minor variations ascribed to individual, biological variability.

The sex ratios were similar in the Control and treated groups, with no statistically significant differences observed.

BODY WEIGHT (OFFSPRING)
There were no effects considered adverse on the offspring weight or weight gain following administration of test item at 62.5, 250 or 1000 mg/kg bw/day to parental generation under the conditions of this study. When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND 0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation. Compared to control, statistical significance was noted in the mean body weights values evaluated for all pups at all dose groups on PND0 (above control) (p<0.01) and on PND4 (above control) (p<0.01) at 250 and 1000 mg/kg bw/day (Mid and High dose). Slightly higher mean body weight gain values were measured for 250 and 1000 mg/kg bw/day (Mid and High dose) between PND0 - 4 (p<0.01). All values were in the normal range, no differences were considered to be related to treatment with test item.

GROSS PATHOLOGY (OFFSPRING)
No macroscopic changes were seen in remaining F1 offspring generation euthanized and examined externally at scheduled termination on PND 4.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

Table 1: Parental (P) generation: Reproductive ability assessment and indices

	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Mated males - females	12/12	12/12	12/12	12/12
Paired males - females	12/12	12/12	12/12	12/12
Infertile males – Non-pregnant females	1/12	1/12	1/12	1/12
Pregnant females, but not delivered	0/11	1/11	0/11	0/11
Male-female Mating Index (%)	100	100	100	100
Male-female Fertility Index (%)	92	92	92	92
Gestation Index (%)	100	91	100	100

Table 2: Parental (P) generation: Gestation, parturition and post-partal period

Females	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Number of delivered dams/dams with implantations	11/11	10/11	11/11	11/11
Duration of pregnancy (days, mean)	22.30	22.80	22.45	22.45
Number of corpora lutea / dams (mean)	15.30	15.90	17.09	17.00
Number of implantations / dams (mean)	14.40	15.00	15.73	15.18
Number of dams with live pups day 0 / number of dams delivered	10/10	10/10	11/11	11/11
Number of dams with live pups day 4 / number of dams delivered	10/10	10/10	11/11	11/11

Table 3: Parental (P) generation: Pre/post-implantation, post-natal and total mortality values (%)

Females	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Pre-implantation mortality (%)	6.77	6.03	8.33	10.41
Intrauterine mortality (%)	12.03	6.99	12.17	10.18
Post-natal mortality (%)	3.33	1.25	1.82	0.00
Total mortality (%)	12.94	7.90	13.18	10.18

Table 4: Offspring (F1) generation: Mortality

	Dose (mg/kg bw/day)
	Control# (10 dams)	62.5# (10 dams)	250 (11 dams)	1000 (11 dams)
Number of pups born (total)	128	140	156	151
Number of pups born (mean)	12.80	14.00	14.18	13.73
Number of dead pups (total) PND0	0	1	3	1
Number of live births (total)	128	140	153	150
Number of live births (mean)	12.80	14.00	13.91	13.64
Number of viable pups (total) PND0	128	139	153	150
Cannibalized pups PND0-4	1	0	1	0
Dead pups (found dead, intact) PND0-4	0	1	3	1
Number of viable pups (total) PND4	127	139	152	150
Number of viable pups (mean) PND4	12.70	13.90	13.82	13.64
Viability indices (%)	99	100	99	100
Not suckled	0	0	0	1
Survival index PND0	100	98.75	96.59	99.43
Survival index PND4	96.67	98.75	95.45	99.43

#: Dams No. 1508 and 2511 excluded from statistics, due to abnormal delivery and not delivered, respectively.

Table 5: Offspring (F1) generation: Sex ratio

 

	Dose (mg/kg bw/day)
	Control# (10 dams)	62.5# (10 dams)	250 (11 dams)	1000 (11 dams)
Sex ratio PND0	43.50	45.85	47.13	52.78
Sex ratio PND4	46.83	45.85	48.49	52.78

#: Dams No. 1508 and 2511 excluded from statistics, due to abnormal delivery and not delivered, respectively.

Table 6: Offspring (F1) generation: Body weights

	Dose (mg/kg bw/day)
	Control	62.5	250	1000
Litter mean:
Mean body weight (g), PND0	10 litters	10 litters	11 litters	11 litters
	6.18	6.66	6.65	6.70	NS
Mean body weight (g), PND4	10 litters	10 litters	11 litters	11 litters
	10.43	11.07	11.31	11.54	NS
Body weight gain (g), PND0-4	4.21	4.40	4.66	5.40	NS
Mean Litter Weight PND0	81.1	93.2	94.1	91.7
Mean Litter Weight PND4	136.8	152.0	156.8	156.6
Mean for all pups:
Mean body weight (g), PND0	128 pups	140 pups	156 pups	151 pups
	6.34	6.66**	6.64**	6.68**	U
Mean body weight (g), PND4	127 pups	139 pups	152 pups	150 pups
	10.77	10.94	11.35**	11.49**	U
Body weight gain (g), PND0-4	4.41	4.27	4.71**	5.42**	U

 ** = p<0.01;NS = not significant ; U = Mann- Whitney U-test versus Control

 

Conclusions:

Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test substance for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.

Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was conducted according to OECD guideline 422. Male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD5, at the following dose levels: 0, 62.5, 250 and 1000 mg/kg bw/day. The measured concentration of the test item evaluated for each test item-dose group varied between 97.9 and 101.5%. No test item was detected in the control samples. These results were within acceptable ranges (90 - 110 %). Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological and ophthalmoscopic assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. Pups were examined at euthanasia. At termination of the adults, necropsy with macroscopic examination was performed; weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives. For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups. Daily administration of the test item by oral gavage to Wistar rats did not result in test item related mortality or clinical adverse effects at daily, weekly or neurological assessment, in ophthalmological changes, or changes in the body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters at dose levels of 62.5, 250, or 1000 mg/kg bw/day during the treatment period under the conditions of this study. No test item related, or adverse effects were noted at evaluation of the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5, under the conditions of this study. There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or at observations following euthanasia. There were no test item-related changes observed in organ weights, at necropsy or at histopathology for the adult animals of either sex. Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test substance for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1 and the study was carried out in accordance with internationally valid GLP principles.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study: OECD guideline. GLP study.

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was conducted according to OECD guideline 422. Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test substance for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day

Short description of key information:
Key study: OECD guideline. GLP study.
A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was conducted according to OECD guideline 422. Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test substance for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Description of key information

Key study: OECD guideline. GLP study. 
A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was conducted according to OECD guideline 422. Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test substance for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1 and the study was carried out in accordance with internationally valid GLP principles.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study: OECD guideline. GLP study.

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was conducted according to OECD guideline 422. Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test substance for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

Based on the available data, the substance is not classified as toxic for reproduction.