Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Subacute NOAEL (reproduction, parental): 120 mg/kg bw/day (OECD 422/GLP)

Subacute NOAEL (development, offspring): 30 mg/kg bw/day (OECD 422/GLP)

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-10-2017 to 23-11-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Species:

rat

Strain:

other: Wistar CRL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: males, females: sexually adult, 7 - 9 weeks on arrival ; dose-range finding experiment: 9 weeks on arrival
- Housing: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage. sterilized soft wood fibers Lignocel.
- Diet: maintenance pelleted diet for rats and mice - Altromin for rats/mice ad libitum, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany (Batch No. 151118/0654, exp.15.11.2018)
- Water: drinking water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
Preparation of the application form was based on the result of the study AAG_467-17-19HS (Annex 1 of this final report). The test item was weighted into a glass beaker and the beaker was replenished by olive oil. The test solution was stirred by magnetic stirrer (500 rpm) for 30 minutes. The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test item to animals was performed during one hour after preparation of application form. The stirring of solutions continued during administration.

Details on mating procedure:

Animals were mated from the 29th day of study. Mating 1:1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Parental males and satellite animals (males and females): 49 days
Parential females: until the end of the lactation period

Frequency of treatment:

Daily

Details on study schedule:

All animals are administered the test item two weeks before mating, during mating, during the pregnancy and lactation period. Parental males and satellite animals (males and females) have a total of 49 days of administration; the parental females are administered until the end of the lactation period (until the 13th post-natal day of pups). Parental females have minimally 49 days of administration, but usually it is a few days longer - depending on which day they become pregnant. In this study the maximum for females was 53-54-52-53 days (0-30-60-120 mg/kg bw/day).

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Control: 12 males + 12 females
Low dose: 12 males + 12 females
Intermediate dose: 12 males + 12 females
High dose: 12 males + 12 females

Satellite groups:
Control: 12 males + 12 females
High dose: 12 males + 12 females

Refer to Table 36. All twelve males and females per group are a part of the Reproduction part of study and examined with the respect to reproduction parameters (biometry of reproduction organs, sperms analysis, and examination of uteri, pups and litters parameters). Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects up to 14 days post treatment.

Control animals:

yes, concurrent vehicle

Details on study design:

STUDY DESIGN
- Dose selection rationale: Due to substantial deaths, two dose-range finding experiments in dose levels 1000, 500, 250, 125 mg/kg/day and 160, 80, 40, 20 mg/kg day had to be performed (Annex 2).

Deaths occurred during the Dose-range finding experiments with the test item Allyl Amyl Glycolate. All males and females at the dose levels 1000, 500 and 250 mg/kg/day died or had to be euthanized due to moribund condition till the third day of experiment. One male died at the dose level 160 mg/kg/day. No death was recorded in animals at the dose level 125, 80, 40 and 20 mg/kg/day.

Clinical symptoms of intoxication as piloerection, hunched posture, apathy, motionless, bradypnoea and decreased reaction to stimuli were recorded in males and females at the dose levels 1000, 500, 250, 125, 160 mg/kg/day.

The lowest body weight of treated animals were recorded in males of the dose levels 125 and 160 mg/kg/day and in females at the dose level 160 mg/kg/day.

The administration of the test item caused slight decrease of total erythrocytes count in males and females at the dose level 160 mg/kg/day compared to other dose levels.

The serious macroscopic changes were observed during necropsy of treated animals. Damage of liver was observed in males and females at the dose levels 1000, 500, 250, 125 and 160 mg/kg/day (males only). Pathological examination of males and females revealed changes of colour and multiple focal changes of liver, enlarged spleen, congested mucosa of small intestine and urinary bladder full of dark red liquid. Males were affected by the test item treatment more than females.

On the basis of the results given above the following dose levels – 120, 60 and 30 mg/kg/day were proposed for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with the test item Ally Amyl Glycolate.


- Rationale for animal assignment (if not random): All twelve males and females per group are a part of the Reproduction part of study and examined with the respect to reproduction parameters (biometry of reproduction organs, sperms analysis, and examination of uteri, pups and litters parameters).Satellite animals are used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effect, for 14 days post treatment.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
Health Condition Control
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before application, during application and immediately after application.

Clinical Observations of Parental Males and Females
All rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00 p.m.). Animals were observed in natural conditions in their cages.

Detailed Clinical Parental Observation
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, breathing, tonic or clonic movements, stereotypes or bizarre behaviour. The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and satellite animals - the first day of administration and then weekly,
Females - the first day of administration and then weekly,
During pregnancy: 0., 7th, 14th, 20th day,
During lactation: 1st, 4th day, 12th day and 13th day
Pups (litters) - 1st, 4th day, 12th day and 13th day
Pups – individually – 4th day of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE:
Main study: Weekly and on the same days as body weight (except the mating period)
Satellite males and females – weekly

CLINICAL CHEMISTRY: Yes (Table 4)
Pups – on the 13th day of lactation for serum levels of thyroid hormone thyroxine (T4 total)
2 pups per litter - on the 4th day of lactation

Oestrous cyclicity (parental animals):

Examination of Vaginal Smears
Vaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of oestrous cycle of females. Only females with regular cyclicity were put into the study. Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa. Vaginal smears have been made also on necropsy day to determine the stage of oestrous cycle

Sperm parameters (parental animals):

In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology.

Sperm Motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm Morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck - were recorded.

Litter observations:

PARAMETERS EXAMINED
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 1 post-partum) and on the 4th day of lactation.

Anogenital Distance (AGD) Measurement
The AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalised to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight.

Nipples Examination
The presence and number of nipples in male pups were counted on day 13 of lactation.

Blood samples from the day 13 pups and the parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total) by ELISA kit


GROSS EXAMINATION OF DEAD PUPS:
The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Postmortem examinations (parental animals):

SACRIFICE
At the end of study, the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of the testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland (all animals) were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight. From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

GROSS PATHOLOGY: Yes
Pathological examination: males and nonpregnant females – after the end of application period; parental females – on the 13th day of lactation; satellite animals – after the end of observation period
Weight of organs: during necropsy

HISTOPATHOLOGY: Yes (Table 5). Full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Organs with macroscopical changes were examined also at the lowest and middle dose level groups. Detailed histological examination of testes was performed with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Statistics:

Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group. The results statistically significant on probability level (p ≤ 0.05) were indicated in the summary tables.

In general:
The parametric tests were used for statistical evaluation of
•body weight of males and females
•mean pup weight
•litter weight
•anogenital distance of pups
•selected haematology parameters
•blood biochemistry parameters
•data from urinalysis (pH, volume)
•data from biometry of organs

As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.

For normally distributed data have at first the variance check has been performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level p ≤ 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences, the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).

The non-parametric tests were used for statistical evaluation of
•selected reproduction parameters with non-continuous distribution (number of live born pups, number of pups, number of implantations)
•selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count)

The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level p ≤ 0.05).

Reproductive indices:

Male mating index
Female mating index
Male fertility index
Female fertility index
Gestation index

Post-implantation loss
Post-natal loss

Offspring viability indices:

Viability index on PND4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The health condition control results were noted for all 12 animals per group and satellite animals (Table Nos. 17 - 22).

Males
The behaviour, clinical status and activity of males in the dose groups 30 and 60 mg/kg bw/day were similar during the study and not different from males of the control group. Mild changes were found during examination of hair (piloerection) and salivation was noted in males at the dose level 120 mg/kg bw/day.

Satellite males
Mild changes persisted in satellite treated males at the dose level 120 mg/kg bw/day. During the examination of hair, piloerection was noted and salivation was recorded.

Females
The behaviour, clinical status and activity of females at the dose levels 30 and 60 mg/kg bw/day were similar during the study and not different from females of the control group. Mild changes were found during examination of hair (piloerection) and salivation was noted in females at the dose level 120 mg/kg bw/day.

Satellite females
Mild changes were found in one female at the dose level 120 mg/kg bw/day. During the examination of hair (piloerection), and salivation, excretion (diarrhoea), reaction to handling (decreased reaction to stimuli) were recorded. This response is unlikely to be related to treatment with the test item, because lesions related to improper administration of the test item (intubation error) were noted in the microscopic examination of this female rat No. 193.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no male parental mortalities. Female No. 163 was found dead on the 12th day of lactation (the reason of death was not detected, because of partial autolysis of organs).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of treated males at the dose level 120 mg/kg bw/day was slightly decreased in comparison to the control males, as shown by the total weight gain. Body weight increments were similar in control and treated males for the whole study (excluding the decrease at the end of study in males at the dose level 120 mg/kg bw/day). Statistically significant differences in necropsy body weight were not found in treated males (Table 38).

Body weights of treated females were comparable among the treated groups but decreased in comparison with the control group of females in the pre-mating period. Weight increments were variable. Mean body weight of all treated groups was decreased in comparison with controls during pregnancy. A statistically significant decrease in body weight was recorded in all treated groups of females on day 20 of pregnancy. Weight increments of treated females at the dose level 120 mg/kg bw/day was lower at the end of the pregnancy period in comparison with the control females. Mean body weight of all treated groups was decreased (on day 4 and 12 of lactation statistically significantly, with the dose dependency) in comparison with controls. Weight increments of treated mothers at the dose level 120 mg/kg bw/day were lower at the end of lactation period in comparison with the control females. (Table 44).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No marked differences were observed between treated and control males in food consumption (Table 39).

The mean food consumption of treated females during the first week was slightly lower in comparison with the control group in the pre-mating period. During the second week, the food consumption was lower in females at the dose level 30 mg/kg bw/day. The mean food consumption of pregnant females treated by the test item was slightly lower (except in first week of pregnancy at the dose level 120 mg/kg bw/day) in comparison with the control group. The statistically significantly decrease of food consumption was recorded in females at the dose level 30 and 60 mg/kg bw/day. The mean food consumption of treated mothers was lower in comparison with the control group of females during lactation. The statistically significantly decrease of food consumption was recorded in females at the dose level 60 and 120 mg/kg bw/day (Table 45).

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:

effects observed, treatment-related

Description (incidence and severity):

Blood (serum) samples from all adult parental males were assessed for thyroid hormone thyroxine (T4 total). Mean concentrations of T4 hormone at the dose levels 60 and 120 mg/kg bw/day were statistically significantly decreased in comparison with control (without dose dependency; Table 42).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Full histopathology of the preserved organs and tissues was performed for high dose and control animals. As there were no treatment-related changes in reproductive organs at the highest dose level, only organs with macroscopic changes and the liver and spleen were examined from the middle and the lowest dose levels (Table 43 and 47). The incidence is expressed in numeric form and ranges in sequence of the dose levels 0-120 mg/kg bw/day.

For examination of the reproduction toxicity of the test item in males, microscopic examination of the testes, epididymis, seminal vesicles, prostate gland, coagulation glands, thyroid gland and pituitary gland was performed for all males of control (No 1-12) and high dose (No 61-72) with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure in the first instance. Histological examination of macroscopically changed organs only was performed also in males at the middle (No 41-52) levels in the second step. No macroscopical findings were recorded in males at the lowest dose level.

Microscopical examination of reproductive organs and the pituitary gland did not reveal presence of treatment-related changes (therefore examination of reproductive organs was performed only in control and high dosed males); only spontaneous changes were noted in males. In the testes, the following microscopic change was recorded: tubular atrophy in 1-0 males. In the prostate gland of 6-0 males, chronic inflammation was observed.

Toxicity of the test item was confirmed by the histological findings of liver (macroscopically changed organ). Focal necrosis of liver was recorded in 0-6 males, bile duct hyperplasia was recorded in 0-12 males. Macroscopically changed organs only from males at the middle and the lowest dose levels were also histologically examined. At the middle dose level, macroscopical findings in the liver and/or stomach were recorded in males Nos. 44 and 46. Microscopically, the hemorrhagic necrosis (marked) of the liver and submucosal edema of the stomach with chronic inflammation (marked) were recorded.

For examination of the reproduction toxicity of the test item, microscopic examination of the reproductive organs, pituitary and thyroid gland was performed for high dose females (Nos. 161-172) and control females (Nos. 101-112). As treatment-related changes at the highest dose level in reproductive organs were not found, histopathological examination of macroscopically changed organs only was performed. Macroscopical changes on the liver and heart were recorded only in females Nos. 147 and 152 at the middle dose levels. No macroscopical findings were recorded in females at the lowest dose level.

The changes related to pregnancy were found in both control and treated females: accumulation of siderophages in mesometrium in 12-9 females and hemosiderin in mucosa of uterus in 12-9 females. Lobular hyperplasia of mammary glands was recorded in 9–5 females. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 0-1 females. Toxicity of the test item was confirmed by the histological findings in the liver (target organ). Focal necrosis of the liver was recorded in 0-6 females, bile duct hyperplasia was recorded in 0-9 females. Macroscopically changed organs from females at the middle dose level were also histologically examined. At the middle dose level, macroscopical findings in the liver and/or heart were recorded in females Nos. 147 and 152. Microscopically, brown pigment and vacuolation in the liver in female No. 147 was recorded. Microscopic examination of the heart in female No 152 did not reveal any finding.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 1 female at 120 mg/kg bw/day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

A comparison of sperm motility in the control males and males from treated groups did not show differences. The test item treatment did not affect sperm morphology. Male rats ability to produce sperm that can fertilise eggs was not affected by the test item administration (Table 41).

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The total number of live pups at the first litter check after parturition, on the 4th day and 13th day of lactation was decreased at all dose levels compared to the control group (Tables 48 & 49). A significantly deceased total number of pups was detected in females at the dose level 60 and 120 mg/kg bw/day on day 4 (due to the death of pups after delivery and/or cannibalism). The five stillborn pups were recorded in females at the dose level 120 mg/kg bw/day only. The mean number of live pups at the first litter check after parturition was dose dependently decreased in all treated groups of females in comparison with the control group of females. Slightly more female then male pups per litter were found in the control group and at the dose level 60 mg/kg bw/day. The opposite ratio (more male then female pups) were born at the dose levels 30 and 120 mg/kg bw/day.

Evidence of copulation was found out in all females (Table 54). A decreased number of females achieving pregnancy was recorded at the dose level 60 and 120 mg/kg bw/day. No abortions were recorded. The mean duration of pregnancy was similar in treated and control groups. The mean number of implantations was the lowest in females at the dose level 60 mg/kg bw/day.

The values of mating indexes showed that mating was not negatively affected by the test item treatment (Table 55). Fertility indexes were lower at the dose level 60 mg/kg bw/day. The gestation index was comparable among the control and treated groups, but viability index on PND 4 was dose-dependently decreased. The lowest viability index was recorded at the dose level 120 mg/kg bw/day. Post-implantation and post-natal losses were significantly increased in females at the dose level 120 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no effects

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of live born pups were significantly changed in treated females in comparison with the control females (Table 54). A statistically significant decreased number of live born pups and number of pups at 4th day of lactation were recorded in females at the dose level 120 and 60 mg/kg bw/day. The stillborns were found only at the dose level 120 mg/kg bw/day. The mean weight of litters at birth, at PND 4 (postnatal day) and PND 13 was statistically significantly decreased at 120 and 60 mg/kg bw/day. The measurement of the AGD (corrected) in pups showed no difference between males and females and was similar in all groups. Probably treatment-related findings were observed in pups at macroscopical examination. In pups at the dose level 120 mg/kg bw/day, the death of pups was found more frequent than in control pups. Some of pups at the dose level 120 mg/kg bw/day were smaller, thinner with little hair in comparison with the control pups. A high incidence of cannibalism (0-0-4-26) was recorded at the dose level 120 mg/kg bw/day. Cannibalism and death of pups were also recorded in females at the dose level 60 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences were recorded (Table 50). The mean body weight of litters and pups at all examination intervals were dose dependently decreased in all treated groups compared to the control group. A statistically significant reduction in the mean body weight of pups was recorded at the dose level 120 mg/kg bw/day; a statistically significant reduction in the mean weight of litters was recorded at the dose levels 60 and 120 mg/kg bw/day during all examination intervals.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Blood samples from the 13-day old pups were assessed for serum levels of thyroid hormone (T4; Table 52). Pup blood was pooled by litter. No statistically significant differences were recorded in the mean concentration of hormone T4 in pups from treated groups against control pups.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The presence of nipples in male pups was checked on day 13 of lactation and none were present (nipple presence in male pups on day 13 of lactation is undesirable).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The weight of the thyroid gland was not statistically significantly changed in male and female pups of treated mothers in comparison with the control group of mothers (Table 53).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The clinical observation of pups was made daily during the study. The macroscopic examination was performed on all surviving pups and/or pups found dead. The examination could not be performed on pups that disappeared due to cannibalism and/or in dead pups with autolysed organs.

Control: 181 of 181 born pups were examined.
Female No. 103: two pups (one male and one female) of the eighteen pups were found dead on the second day after delivery – empty stomach was recorded. No macroscopical findings were recorded in other pups/litters.

30 mg/kg bw/day: 162 of 162 born pups were examined.
Female No. 123: one female pup of the thirteen pups was found dead after delivery (live birth) – no macroscopical findings were recorded. No macroscopical findings were recorded in other pups/litters.

60 mg/kg bw/day: 113 of 117 born pups were examined.
Female No. 147: she lost all fifteen pups (whole litter) by the 4th day of lactation. After delivery, nine live pups (one male + eight females) and six dead pups (three males and three females - they were born alive) were recorded. On the second day only one male pup was alive, five female pups were found dead and three pups disappeared (cannibalism). The remaining male pup disappeared (cannibalism) by the 4th day of lactation. All examined pups - no macroscopical findings were recorded.
Female No. 151: one male pup of the fourteen pups was found dead on the second day after delivery. No macroscopical findings were recorded. No macroscopical findings were recorded in other pups/litters.

120 mg/kg bw/day: 100 of 126 born pups were examined.
Female No. 162: all surviving fifteen pups were markedly smaller, thinner and had less hair. The four pups (two males and two females) disappeared by the seventh day of lactation due to cannibalism therefore they were not examined.
Female No. 163: she was found dead on day 11 of lactation. Pups were alive, but undercooled, thin, with an empty stomach.
Female No. 164: nine (one female and eight males) of eighteen pups disappeared the second day after delivery due to cannibalism. No macroscopical finding were recorded in remaining nine pups.
Female No. 165: a total of ten dead pups were found after delivery. Two of them were born alive; the other two could not be examined due to partial autolysis of organs and the other six could not be examined due to partial cannibalism. Examined pups: no macroscopical findings were recorded.
Female No. 168: fifteen pups were recorded after delivery. Three (two males and one female) of fifteen pups were alive – they were found dead the second day after delivery. Seven pups were found dead after delivery (two males and five females) – they were born alive. Five pups were found dead after delivery – they were born dead (partial cannibalism was observed). Examined pups: no macroscopical findings were recorded.
Female No. 170: one female pup of twelve pups was found dead after delivery. The pup was born alive, no macroscopical findings were recorded.
Female No. 172: all six surviving pups were less hairy.
No macroscopical findings were recorded in other pups/litters.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: increased cannibalism and on macroscopical findings (smaller, thinner pups, death of pups) of some pups at the dose levels 60 and 120 mg/kg bw/day.

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In a combined repeated dose and reproduction/developmental toxicity screening test in Wistar rats, the NOAEL for reproduction was established as 120 mg/kg body weight/day. All changes in reproductive parameters observed in parental males and females at all dose levels were considered to be of no toxicological significance. The NOAEL for development was established as 30 mg/kg body weight/day. This judgement is based predominantly on increased cannibalism and on macroscopical findings (smaller, thinner pups, death of pups) of some pups at the dose levels 60 and 120 mg/kg bw/day.

Executive summary:

In a combined repeated dose and reproduction/developmental toxicity screening test (18-343), Allyl Amyl Glycolate was administered to 4 groups of Wistar rats (12 animals/sex/group) by gavage in olive oil at dose levels of 0, 30, 60 and 120 mg/kg bw/day, 7 days per week, for 49 days (parental males and all satellite animals) and until the end of the lactation period (parental females).

There were no male parental mortalities. One female was found dead on the 12th day of lactation (the reason of death was not detected, because of partial autolysis of organs). Body weight of treated males at the dose level 120 mg/kg bw/day was slightly decreased in comparison to the control males, as shown by the total weight gain. Body weight increments were similar in control and treated males for the whole study (excluding the decrease at the end of study in males at the dose level 120 mg/kg bw/day). Statistically significant differences in necropsy body weight were not found in treated males. Body weights of treated females were comparable among the treated groups but decreased in comparison with the control group of females in the pre-mating period. Weight increments were variable. Mean body weight of all treated groups was decreased in comparison with controls during pregnancy. A statistically significant decrease in body weight was recorded in all treated groups of females on day 20 of pregnancy. Weight increments of treated females at the dose level 120 mg/kg bw/day was lower at the end of the pregnancy period in comparison with the control females.  Mean body weight of all treated groups was decreased (on day 4 and 12 of lactation statistically significantly, with the dose dependency) in comparison with controls. Weight increments of treated mothers at the dose level 120 mg/kg bw/day were lower at the end of lactation period in comparison with the control females. No marked differences were observed between treated and control males in food consumption. The mean food consumption of treated females during the first week was slightly lower in comparison with the control group in the pre-mating period. During the second week, the food consumption was lower in females at the dose level 30 mg/kg bw/day. The mean food consumption of pregnant females treated by the test item was slightly lower (except in first week of pregnancy at the dose level 120 mg/kg bw/day) in comparison with the control group. The statistically significantly decrease of food consumption was recorded in females at the dose level 30 and 60 mg/kg bw/day. The mean food consumption of treated mothers was lower in comparison with the control group of females during lactation. The statistically significantly decrease of food consumption was recorded in females at the dose level 60 and 120 mg/kg bw/day.

Blood (serum) samples from all adult parental males were assessed for thyroid hormone thyroxine (T4 total).  Mean concentrations of T4 hormone at the dose levels 60 and 120 mg/kg bw/day were statistically significantly decreased in comparison with control (without dose dependency). Absolute weight of reproductive organs – testes, epididymis and prostate gland with seminal vesicles was slightly decreased in males at 120 mg/kg bw/day in comparison with the control group of males. Absolute weight of pituitary gland was insignificantly decreased in males at the dose level 60 mg/kg compared to control males.  Relative weight of reproductive organs – testes, epididymis and prostate gland with seminal of treated groups of males was comparable to the control males. The relative weight of the pituitary gland of treated males was similar to the control males.  Relative weight of thyroid gland was statistically significantly increased in males at the dose level 120 mg/kg bw/day. Examination of absolute weight of reproductive organs – the ovaries were decreased in weight in all treated groups; (p ≤0.05 at dose levels 30 and 60 mg/kg bw/day). The absolute weight of the uterus in treated groups were similar to the control group. The absolute weight of the pituitary gland was significantly decreased in females at the dose levels 60 and 120 mg/kg bw/day mg/kg compared to control females. Examination of relative weight of reproductive organs – the ovaries were decreased in weight compared to the control females but it was not statistically significant. The relative weight of the pituitary gland of treated females was decreased (p ≤0.05 at dose level 60 mg/kg). The absolute and relative weight of the thyroid gland of treated females was comparable to the control females.

A comparison of sperm motility in the control males and males from treated groups did not show differences. The test item treatment did not affect sperm morphology. Male rats ability to produce sperm that can fertilise eggs was not affected by the test item administration. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 1 female at 120 mg/kg bw/day.

No treatment-related macroscopical changes in the reproductive organs, pituitary and thyroid glands were noted in treated males or females.  Full histopathology of the preserved organs and tissues was performed for high dose and control animals. As there were no treatment-related changes in reproductive organs at the highest dose level, only organs with macroscopic changes and the liver and spleen were examined from the middle and the lowest dose levels. The incidence is expressed in numeric form and ranges in sequence of the dose levels 0-120 mg/kg bw/day. No macroscopical findings were recorded in males at the lowest dose level. Microscopical examination of reproductive organs and the pituitary gland did not reveal presence of treatment-related changes (therefore examination of reproductive organs was performed only in control and high dosed males); only spontaneous changes were noted in males. In the testes, the following microscopic change was recorded: tubular atrophy in 1-0 males. In the prostate gland of 6-0 males, chronic inflammation was observed. Toxicity of the test item was confirmed by the histological findings of liver (macroscopically changed organ). Focal necrosis of liver was recorded in 0-6 males, bile duct hyperplasia was recorded in 0-12 males. Macroscopically changed organs only from males at the middle and the lowest dose levels were also histologically examined. At the middle dose level, macroscopical findings in the liver and/or stomach were recorded in 2 males. Microscopically, the hemorrhagic necrosis (marked) of the liver and submucosal edema of the stomach with chronic inflammation (marked) were recorded.

As treatment-related changes at the highest dose level in reproductive organs were not found, histopathological examination of macroscopically changed organs only was performed. Macroscopical changes on the liver and heart were recorded only in 2 females at the middle dose level. No macroscopical findings were recorded in females at the lowest dose level. The changes related to pregnancy were found in both control and treated females: accumulation of siderophages in mesometrium in 12-9 females and hemosiderin in mucosa of uterus in 12-9 females. Lobular hyperplasia of mammary glands was recorded in 9–5 females. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 0-1 females. Toxicity of the test item was confirmed by the histological findings in the liver (target organ). Focal necrosis of the liver was recorded in 0-6 females, bile duct hyperplasia was recorded in 0-9 females. Macroscopically changed organs from females at the middle dose level were also histologically examined. At the middle dose level, macroscopical findings in the liver and/or heart were recorded in 2 females. Microscopically, brown pigment and vacuolation in the liver in 1 female was recorded. Microscopic examination of the heart in 1 female did not reveal any finding.

Evidence of copulation was found out in all females. A decreased number of females achieving pregnancy was recorded at the dose level 60 and 120 mg/kg bw/day.  No abortions were recorded. The mean duration of pregnancy was similar in treated and control groups.  The mean number of implantations was the lowest in females at the dose level 60 mg/kg bw/day.The values of mating indexes showed that mating was not negatively affected by the test item treatment. Fertility indexes were lower at the dose level 60 mg/kg bw/day. The gestation index was comparable among the control and treated groups, but viability index on PND 4 was dose-dependently decreased. The lowest viability index was recorded at the dose level 120 mg/kg bw/day. Post-implantation and post-natal losses were significantly increased in females at the dose level 120 mg/kg bw/day.

The number of live born pups were significantly changed in treated females in comparison with the control females. A statistically significant decreased number of live born pups and number of pups at 4th day of lactation were recorded in females at the dose level 120 and 60 mg/kg bw/day. The stillborns were found only at the dose level 120 mg/kg bw/day. The mean weight of litters at birth, at PND 4 (postnatal day) and PND 13 was statistically significantly decreased at 120 and 60 mg/kg bw/day. Statistically significant differences were recorded in body weights. The mean body weight of litters and pups at all examination intervals were dose dependently decreased in all treated groups compared to the control group. A statistically significant reduction in the mean body weight of pups was recorded at the dose level 120 mg/kg bw/day; a statistically significant reduction in the mean weight of litters was recorded at the dose levels 60 and 120 mg/kg bw/day during all examination intervals. The presence of nipples in male pups was checked on day 13 of lactation and none were present (nipple presence in male pups on day 13 of lactation is undesirable). The measurement of the AGD (corrected) in pups showed no difference between males and females and was similar in all groups. The body weight at measuring of the AGD)(on the 4th day of lactation) was statistically significantly decreased in male and female pups at the dose level 120 mg/kg bw/day. Corrected anogenital distance did not show significant changes among the treated groups and control group. No differences in the postnatal development of pups were observed in the control and treated groups. Blood samples from the 13-day old pups were assessed for serum levels of thyroid hormone (T4). Pup blood was pooled by litter. No statistically significant differences were recorded in the mean concentration of hormone T4 in pups from treated groups against control pups. The weight of the thyroid gland was not statistically significantly changed in male and female pups of treated mothers in comparison with the control group of mothers.

The total number of pups and their mean body weights at first litter check after parturition and during the next intervals were significantly decreased in females at the dose levels 120 and 60 mg/kg bw/day.  Stillborn pups were detected in females at the dose level 120 mg/kg bw/day only. Macroscopical examination of pups showed an increased number with macroscopical findings at the dose level 120 mg/kg bw/day. Markedly smaller, thinner and less hairy pups were noted in two litters. An increased incidence of cannibalism and death of the pups was observed in females at the dose level 60 and 120 mg/kg bw/day. Twenty-six pups at the dose level 120 mg/kg bw/day and 4 pups at the dose levels 60 mg/kg bw/day could not be examined due to partial or total cannibalism. Thirteen pups at the dose level 120 mg/kg bw/day and 12 pups at the dose level 60 mg/kg bw/day were found dead during the lactation period. This effect – cannibalism, was not observed in the control group and the lowest group. Cannibalism was recorded sporadically at 60 mg/kg bw/day and markedly at 120 mg/kg bw/day. Therefore, dose dependency was recorded. Simultaneously it was found that mothers who cannibalized some pups were also good carers of their remaining pups. It could be speculated that only the altered pups were subject to cannibalism and that this phenomenon could therefore be used as indirect evidence that in litters affected by cannibalism, some type of developmental toxicity effects caused by the test substance treatment were present. On the other hand, the possibility that cannibalism could be a consequence of the test item toxicity to mothers cannot be ignored. Decreased food consumption and decreased body weight of mothers of the dose levels 60 and 120 mg/kg bw/day were recorded. Mothers who are stressed may show bizarre behaviour, including cannibalism. The toxic effect of the test item via lactation is unlikely because at the highest dose of 120 mg/kg bw/day there were also females in which cannibalism and/or death of pups were not recorded and the offspring were bred.

The NOAEL for reproduction was established as 120 mg/kg body weight/day. All changes in reproductive parameters observed in parental males and females at all dose levels were considered to be of no toxicological significance. The NOAEL for development was established as 30 mg/kg body weight/day. This judgement is based predominantly on increased cannibalism and on macroscopical findings (smaller, thinner pups, death of pups) of some pups at the dose levels 60 and 120 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was an OECD 422/GLP study and is the only study available. It was assigned a Klimisch score of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is one combined repeated dose and reproduction/developmental toxicity screening test in rats available.

In a combined repeated dose and reproduction/developmental toxicity screening test (18-343), Allyl Amyl Glycolate was administered to 4 groups of Wistar rats (12 animals/sex/group) by gavage in olive oil at dose levels of 0, 30, 60 and 120 mg/kg bw/day, 7 days per week, for 49 days (parental males and all satellite animals) and until the end of the lactation period (parental females).

There were no male parental mortalities. One female was found dead on the 12th day of lactation (the reason of death was not detected, because of partial autolysis of organs). Body weight of treated males at the dose level 120 mg/kg bw/day was slightly decreased in comparison to the control males, as shown by the total weight gain. Body weight increments were similar in control and treated males for the whole study (excluding the decrease at the end of study in males at the dose level 120 mg/kg bw/day). Statistically significant differences in necropsy body weight were not found in treated males. Body weights of treated females were comparable among the treated groups but decreased in comparison with the control group of females in the pre-mating period. Weight increments were variable. Mean body weight of all treated groups was decreased in comparison with controls during pregnancy. A statistically significant decrease in body weight was recorded in all treated groups of females on day 20 of pregnancy. Weight increments of treated females at the dose level 120 mg/kg bw/day was lower at the end of the pregnancy period in comparison with the control females.  Mean body weight of all treated groups was decreased (on day 4 and 12 of lactation statistically significantly, with the dose dependency) in comparison with controls. Weight increments of treated mothers at the dose level 120 mg/kg bw/day were lower at the end of lactation period in comparison with the control females. No marked differences were observed between treated and control males in food consumption. The mean food consumption of treated females during the first week was slightly lower in comparison with the control group in the pre-mating period. During the second week, the food consumption was lower in females at the dose level 30 mg/kg bw/day. The mean food consumption of pregnant females treated by the test item was slightly lower (except in first week of pregnancy at the dose level 120 mg/kg bw/day) in comparison with the control group. The statistically significantly decrease of food consumption was recorded in females at the dose level 30 and 60 mg/kg bw/day. The mean food consumption of treated mothers was lower in comparison with the control group of females during lactation. The statistically significantly decrease of food consumption was recorded in females at the dose level 60 and 120 mg/kg bw/day.

Blood (serum) samples from all adult parental males were assessed for thyroid hormone thyroxine (T4 total).  Mean concentrations of T4 hormone at the dose levels 60 and 120 mg/kg bw/day were statistically significantly decreased in comparison with control (without dose dependency). Absolute weight of reproductive organs – testes, epididymis and prostate gland with seminal vesicles was slightly decreased in males at 120 mg/kg bw/day in comparison with the control group of males. Absolute weight of pituitary gland was insignificantly decreased in males at the dose level 60 mg/kg compared to control males.  Relative weight of reproductive organs – testes, epididymis and prostate gland with seminal of treated groups of males was comparable to the control males. The relative weight of the pituitary gland of treated males was similar to the control males.  Relative weight of thyroid gland was statistically significantly increased in males at the dose level 120 mg/kg bw/day. Examination of absolute weight of reproductive organs – the ovaries were decreased in weight in all treated groups; (p ≤0.05 at dose levels 30 and 60 mg/kg bw/day). The absolute weight of the uterus in treated groups were similar to the control group. The absolute weight of the pituitary gland was significantly decreased in females at the dose levels 60 and 120 mg/kg bw/day mg/kg compared to control females. Examination of relative weight of reproductive organs – the ovaries were decreased in weight compared to the control females but it was not statistically significant. The relative weight of the pituitary gland of treated females was decreased (p ≤0.05 at dose level 60 mg/kg). The absolute and relative weight of the thyroid gland of treated females was comparable to the control females.

A comparison of sperm motility in the control males and males from treated groups did not show differences. The test item treatment did not affect sperm morphology. Male rats ability to produce sperm that can fertilise eggs was not affected by the test item administration. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 1 female at 120 mg/kg bw/day.

No treatment-related macroscopical changes in the reproductive organs, pituitary and thyroid glands were noted in treated males or females.  Full histopathology of the preserved organs and tissues was performed for high dose and control animals. As there were no treatment-related changes in reproductive organs at the highest dose level, only organs with macroscopic changes and the liver and spleen were examined from the middle and the lowest dose levels. The incidence is expressed in numeric form and ranges in sequence of the dose levels 0-120 mg/kg bw/day. No macroscopical findings were recorded in males at the lowest dose level. Microscopical examination of reproductive organs and the pituitary gland did not reveal presence of treatment-related changes (therefore examination of reproductive organs was performed only in control and high dosed males); only spontaneous changes were noted in males. In the testes, the following microscopic change was recorded: tubular atrophy in 1-0 males. In the prostate gland of 6-0 males, chronic inflammation was observed. Toxicity of the test item was confirmed by the histological findings of liver (macroscopically changed organ). Focal necrosis of liver was recorded in 0-6 males, bile duct hyperplasia was recorded in 0-12 males. Macroscopically changed organs only from males at the middle and the lowest dose levels were also histologically examined. At the middle dose level, macroscopical findings in the liver and/or stomach were recorded in 2 males. Microscopically, the hemorrhagic necrosis (marked) of the liver and submucosal edema of the stomach with chronic inflammation (marked) were recorded.

As treatment-related changes at the highest dose level in reproductive organs were not found, histopathological examination of macroscopically changed organs only was performed. Macroscopical changes on the liver and heart were recorded only in 2 females at the middle dose level. No macroscopical findings were recorded in females at the lowest dose level. The changes related to pregnancy were found in both control and treated females: accumulation of siderophages in mesometrium in 12-9 females and hemosiderin in mucosa of uterus in 12-9 females. Lobular hyperplasia of mammary glands was recorded in 9–5 females. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 0-1 females. Toxicity of the test item was confirmed by the histological findings in the liver (target organ). Focal necrosis of the liver was recorded in 0-6 females, bile duct hyperplasia was recorded in 0-9 females. Macroscopically changed organs from females at the middle dose level were also histologically examined. At the middle dose level, macroscopical findings in the liver and/or heart were recorded in 2 females. Microscopically, brown pigment and vacuolation in the liver in 1 female was recorded. Microscopic examination of the heart in 1 female did not reveal any finding.

Evidence of copulation was found out in all females. A decreased number of females achieving pregnancy was recorded at the dose level 60 and 120 mg/kg bw/day.  No abortions were recorded. The mean duration of pregnancy was similar in treated and control groups.  The mean number of implantations was the lowest in females at the dose level 60 mg/kg bw/day.The values of mating indexes showed that mating was not negatively affected by the test item treatment. Fertility indexes were lower at the dose level 60 mg/kg bw/day. The gestation index was comparable among the control and treated groups, but viability index on PND 4 was dose-dependently decreased. The lowest viability index was recorded at the dose level 120 mg/kg bw/day. Post-implantation and post-natal losses were significantly increased in females at the dose level 120 mg/kg bw/day.

The number of live born pups were significantly changed in treated females in comparison with the control females. A statistically significant decreased number of live born pups and number of pups at 4th day of lactation were recorded in females at the dose level 120 and 60 mg/kg bw/day. The stillborns were found only at the dose level 120 mg/kg bw/day. The mean weight of litters at birth, at PND 4 (postnatal day) and PND 13 was statistically significantly decreased at 120 and 60 mg/kg bw/day. Statistically significant differences were recorded in body weights. The mean body weight of litters and pups at all examination intervals were dose dependently decreased in all treated groups compared to the control group. A statistically significant reduction in the mean body weight of pups was recorded at the dose level 120 mg/kg bw/day; a statistically significant reduction in the mean weight of litters was recorded at the dose levels 60 and 120 mg/kg bw/day during all examination intervals. The presence of nipples in male pups was checked on day 13 of lactation and none were present (nipple presence in male pups on day 13 of lactation is undesirable). The measurement of the AGD (corrected) in pups showed no difference between males and females and was similar in all groups. The body weight at measuring of the AGD (on the 4th day of lactation) was statistically significantly decreased in male and female pups at the dose level 120 mg/kg bw/day. Corrected anogenital distance did not show significant changes among the treated groups and control group. No differences in the postnatal development of pups were observed in the control and treated groups. Blood samples from the 13-day old pups were assessed for serum levels of thyroid hormone (T4). Pup blood was pooled by litter. No statistically significant differences were recorded in the mean concentration of hormone T4 in pups from treated groups against control pups. The weight of the thyroid gland was not statistically significantly changed in male and female pups of treated mothers in comparison with the control group of mothers.

The total number of pups and their mean body weights at first litter check after parturition and during the next intervals were significantly decreased in females at the dose levels 120 and 60 mg/kg bw/day.  Stillborn pups were detected in females at the dose level 120 mg/kg bw/day only. Macroscopical examination of pups showed an increased number with macroscopical findings at the dose level 120 mg/kg bw/day. Markedly smaller, thinner and less hairy pups were noted in two litters. An increased incidence of cannibalism and death of the pups was observed in females at the dose level 60 and 120 mg/kg bw/day. Twenty-six pups at the dose level 120 mg/kg bw/day and 4 pups at the dose levels 60 mg/kg bw/day could not be examined due to partial or total cannibalism. Thirteen pups at the dose level 120 mg/kg bw/day and 12 pups at the dose level 60 mg/kg bw/day were found dead during the lactation period. This effect – cannibalism, was not observed in the control group and the lowest group. Cannibalism was recorded sporadically at 60 mg/kg bw/day and markedly at 120 mg/kg bw/day. Therefore, dose dependency was recorded. Simultaneously it was found that mothers who cannibalized some pups were also good carers of their remaining pups. It could be speculated that only the altered pups were subject to cannibalism and that this phenomenon could therefore be used as indirect evidence that in litters affected by cannibalism, some type of developmental toxicity effects caused by the test substance treatment were present. On the other hand, the possibility that cannibalism could be a consequence of the test item toxicity to mothers cannot be ignored. Decreased food consumption and decreased body weight of mothers of the dose levels 60 and 120 mg/kg bw/day were recorded. Mothers who are stressed may show bizarre behaviour, including cannibalism. The toxic effect of the test item via lactation is unlikely because at the highest dose of 120 mg/kg bw/day there were also females in which cannibalism and/or death of pups were not recorded and the offspring were bred.

The NOAEL for reproduction was established as 120 mg/kg body weight/day. All changes in reproductive parameters observed in parental males and females at all dose levels were considered to be of no toxicological significance. The NOAEL for development was established as 30 mg/kg body weight/day. This judgement is based predominantly on increased cannibalism and on macroscopical findings (smaller, thinner pups, death of pups) of some pups at the dose levels 60 and 120 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information in the dossier, the conclusion for the substance Allyl Amyl Glycolate  (EC No. 916-328-0) for reproductive toxicity is inconclusive and the substance does not need to be classified for effects on or via lactation when the criteria outlined in Annex I of 1227/2008/EC are applied.