Tributyl O-acetylcitrate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Four groups of 30 male and 30 female Sprague Dawley strain rats were given acetyl tributyl citrate (ATBC) in the diet at dose levels of 0 (control), 100, 300 or 1000 mg/kg/day, continuously for the time they remained part of the study. These were designated F0animals and were fed the diets containing the test article at concentrations calculated from preliminary or historical data, or from estimated food consumptions and body weights. Prior to mating, the males were treated for 77 days, and females for 21 days, when one male was paired with one female from the same treatment group for up to two weeks. After confirmation of mating, the male was removed from the female and fed the relevant diet for a further 3-8 weeks, depending upon the time of mating and scheduled necropsy.

In summary, apart from the lower water intakes of the 300 and 1000 mg/kg body weight/day ATBC treated F0 and F1 generation female rats during pregnancy, and the lower body weights of the F1 male groups fed ATBC at levels of 300 and 1000 mg/kg/day, acetyl tributyl citrate did not cause any reproductive toxicity effects in the rat under the conditions of this two generation study.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study was conducted according to OECD TG 416 and in accordance with the Principles of GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Certain minor deviations were noted during the study condcut and these were deemed not have an adverse impact on the outcome of the study

Deviations:

yes

Remarks:

Certain minor deviations were noted during the study condcut and these were deemed not have an adverse impact on the outcome of the study

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Justification for study design:

not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
The test article, acetyl tributyl citrate (ATBC), was supplied as a clear oily liquid, as four separate batches, one batch from each of the following suppliers and identified as:
1. ATBC, (Batch Not specified), Asahi Chemical Industry Co Ltd Osaka, Japan
2. Citroflex, A-4, N 95083, Krehalon Industrie B.V. Holland
3. Uniplex 84, Lot 82799, Unitex Chemical Corporation N.C. 27406 USA
4. Citroflex, A-4, N98221-H701064M, Morflex Inc. N.C. 27403 USA

- Expiration date of the lot/batch: no information
- Purity test date: no information

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Equal quantities of each batch of the test article were blended together in a large container and distributed into darkened glass bottles for storage at room temperature.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Recommended test species by various regulatory agencies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac Ltd. Bicester, Oxon, UK
- Females nulliparous and non-pregnant: [yes]
- Age at study initiation: Unproven male and nulliparous female rats of the Sprague Dawley strain were obtained at approximately six weeks of age from a barrier reared, virus free colony.
- Fasting period before study: no
- Housing: They were housed in polypropylene cages with stainless-steel grid tops and floors, suspended over paper for the removal of excreta. The rats were housed in groups of two at all times, except during mating, when one male was housed with one female from the same treatment group. After mating, the males were housed as in the pre-mating phase, and the females were housed individually in similar cages, but with sawdust covered solid floors. Paper nesting material was also provided when necessary
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: They were kept in the study room for six days prior to start of treatment to allow them to acclimatize to the environmental conditions
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24 degree Celsius
- Humidity (%): 45-70 %
- Air changes (per hr): approximately 15/hour with no recirculation using high efficieny filetrs
- Photoperiod (hrs dark / hrs light): 12 hours light:dark cycle

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION - The required quantity of ATBC was weighed into a small quantity of diet and mixed using an electric hand mixer for three minutes. This was subsequently blended with two further amounts of control diet, using a paddle mixer and a cone mixer respectively, for a minimum of 15 minutes each, to give the required amount of diet at each concentration. Occasionally, smaller mixes were required which omitted one or both of the last two stages of the diet mixing procedure.
Diets containing ATBC were prepared at two week intervals over the feeding phases of the study. During the pregnancy, lactation and weaning stages it was necessary to prepare some diets at weekly intervals depending on feeding requirements. After each mix, two samples of each diet were analysed for test article concentration and these diet mixes were accepted for use if the mean of the results was within 10% of the intended concentration. On each occasion of analysis, two samples of control diet were analysed for test article content

Details on mating procedure:

- M/F ratio per cage: 1:1 (male:female
- Length of cohabitation: 3 weeks
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- After successful mating each pregnant female was caged (how): individually housed
- Any other deviations from standard protocol: no information on additional mating opportunities after unsuccessful cohabitation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the two concentrations of ATBC prepared for the mixing efficiency and stability studies demonstrated that the mean concentrations were within 10% of the intended concentrations, with coefficients of variance of 1.92 and 2.74% for the 0.05 and 2.0% ATBC diets respectively, indicating that the mixing procedures were adequate. Analysis of diet from open feed pots did not indicate any loss of test article from the diet over the seven day period, and analysis of diets stored in closed stainless steel containers at 2-6°C indicated that the prepared diets were stable for at least 21 days.

One batch of diet fed to the top dose pregnant F, rats was required for use for a few days after its three week expiry date. This was discarded in error
before it was subsequently analysed for test article content. Previous stability data up to day 21 shows no loss of test article and it was considered that this diet would be acceptable for use.

Analysis of the diets containing ATBC demonstrated that the prepared diets that were fed to the rats over the course of the study contained within +/-10% of the intended concentration of ATBC

Duration of treatment / exposure:

Conitunous throughout the study period

Frequency of treatment:

daily exposure via diet throughout the study period

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were between 35 and 48 days of age after birth
- Age at mating of the mated animals in the study approximately 13 weeks

Dose / conc.:

100 mg/kg diet

Remarks:

100 mg/kg/day ATBC

Dose / conc.:

300 mg/kg diet

Remarks:

300 mg/kg/day ATBC

Dose / conc.:

1 000 mg/kg diet

Remarks:

1000 mg/kg/day ATBC

No. of animals per sex per dose:

30 males + 30 females per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no information available
- Rationale for animal assignment (if not random): During the acclimatization period they were assigned to treatment groups using random number techniques and the weight range checked to ensure an even distribution.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in its cage for variation of its appearance or condition, the appearance of its excreta or of its behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly, at the time of a weighing a more detailed examination was made

BODY WEIGHT: Yes
The rats were weighed as follows:
F0 generation - Males
The rats were weighed daily, for three days before to three days after the first day of treatment, and subsequently weekly throughout the treatment and mating periods until killed.
F0 generation - Females
The rats were weighed daily, for three days before to three days after the first day of treatment, and subsequently weekly throughout the treatment and mating periods, or until positive evidence of mating was obtained. They were weighed daily from the day that conclusive evidence of mating was obtained until the day of littering. The weight of each female and the total litter weight was recorded on the day after birth (day 1) and on days 4,7 and 14. On day 21 the female and each of the young were weighed individually. The F0 females were then weighed weekly after weaning until killed.
Females found to be non-pregnant were weighed daily to a maximum of 24 days after separation from the male, and then weekly until killed.
F1 generation - Males
The rats were weighed weekly from the first Monday after weaning until killed.
F1 generation - Females
The rats were weighed weekly from the first Monday after weaning throughout the treatment and mating periods until positive evidence of mating was obtained. They were weighed daily from the day that conclusive evidence of mating was obtained until the day of littering. The weight of each female and the total litter weight was recorded on the day after birth (day 1) and on days 4,7 and 14. On day 21 the female and each of the young (F2 generation) were weighed individually. The F1 females were then weighed weekly after weaning until killed

FOOD CONSUMPTION AND COMPOUND INTAKE AND WATER INTAKE:
The food and water intakes for each cage of F0 and F1 generation rats was measured between the intervals of weighing, at all times except during the period when males and females were housed together for mating and during the weaning period when intakes were measured for females on days 10 and 17, as well as on days 7, 14 and 21 post-partum, when body weights were recorded.
No intakes were recorded for F0 males after study day 77, and intakes for non-pregnant F0 females were not always recorded. It is not considered that these deviations from the study protocol adversely affected the outcome of the study.

OTHER: The day of littering was recorded and the gestation time calculated

Oestrous cyclicity (parental animals):

not evaluated

Sperm parameters (parental animals):

not evaluated

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter,excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was/ determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not evaluated

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not evaluated

Postmortem examinations (parental animals):

Adult animals
Necropsy examinations were performed on the F0 and F1 males and females that produced a litter within 14 days of weaning the litter and on the F0 and F1 males and females that did not produce litters, within 42 days from their last day of mating. This minor deviation from the study protocol defined period of 23-30 days after mating is not considered to have adversely affected the outcome of the study.
The rats were deprived of food overnight and were killed by exsanguination under barbiturate anaesthesia. Any abnormalities observed were recorded.
Samples of the following tissues, according to sex, were retained in 10% buffered formalin:
adipose tissue (perirenal), adrenal glands, artery (aorta), bladder (urinary), bone marrow, brain, caecum, colon, cervix uteri, diaphragm, duodenum, epididyrnis, eye (fixed in Davidsons fluid for 24 hr), femur,Harderian gland, head, heart, ileum, jejunum, kidneys, liver (representative samples from each lobe), lungs (inflated with formalin before removal), lymph nodes (axillary, cervical and mesenteric), mammary gland (inguinal region),
nasal bones, nerve (sciatic taken with surrounding muscle), oesophagus, ovaries, pancreas, pinnae (retained for identification as appropriate), pituitary, prostate, rectum, salivary gland, seminal vesicles, skeletal muscle
skin (inguinal region), spinal cord (retained in vertebral column), spleen, stomach, testes, thymus, thyroid gland (retained on trachea), tongue, trachea, uterine horns, vagina, vein (posterior vena cava), any other abnormal tissue.
Wax embedded sections (approximately 5 microns) were prepared from the tissues found to be abnormal at necropsy. These sections were stained with haematoxylin and eosin and examined microscopically

Postmortem examinations (offspring):

The litters were examined on days 4, 7, 10, 14, 17 and 21 for the number of survivors and any abnormalities, and on day 21 for the sex of each pup. A necropsy examination was performed within 14 days of weaning on each litter born in the F1 and F2 generations, except on those animals selected to produce the F2 generation.
One animal of each sex from each litter, if available, was randomly selected and subjected to a necropsy procedure, including tissue retention, as for adult animals.
The remaining animals in each litter were killed by carbon dioxide asphyxiation and the tissues examined and any abnormalities recorded. Tissues were retained only if they were thought to show pathological abnormalities. Any pups found dead during the study were, if possible, examined similarly. Any tissues retained were stored in neutral buffered formalin.
Wax embedded sections (approximately 5 microns) were prepared from the tissues found to be abnormal at necropsy. These sections were stained with haematoxylin and eosin and examined microscopically

Statistics:

Standard statistical methods were employed

Reproductive indices:

not calculated

Offspring viability indices:

not calculated

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Observations of the condition and behaviour of the F0 generation rats during their time on the study were as -
There were a few incidences where cutaneous and subcutaneous lumps were observed in the genital areas of F0 male rats. These were generally black in colour, 5-10 mm in diameter with an incidence of 1/30, 1/30, 4/30 and 4/30 for the 0,100,300 and 1000 mg/kg/day ATBC groups respectively. At the necropsy examination, this finding was seen in further rats (6.10.), and was distributed fairly evenly throughout all groups, including the controls. These observations were not made in any of the F0 female rats. There were also a few (2/30) incidences of red staining around the eyes in each of the F0 male groups fed ATBC. This observation was limited to one F0 female rat in the 300 mg/kg/day treatment group. Another animal in this group was found to have a bruised and swollen left hind foot at necropsy. Staining around the genital area was also observed in one female F0 rat fed ATBC at a level of 1000 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F0 generation - Males
There were no obvious differences in the mean body weights of the F0 generation male rats fed ATBC at dose levels of 100,300 or 1000 mg/kg/day
over a 17 week period, when compared with control values.
F0 generation - Females
There were no noticeable differences in the mean body weights of the F0 generation female rats fed ATBC in the diet at dose levels of 100 or 300 mg/kg/day over the three week pre mating period. The mean bodyweight of the group fed ATBC at a level of 1000 mg/kg /day was statistically significantly lower than the control value only on day 1 of treatment.
The mean body weights of the groups fed ATBC were largely unaffected by treatment throughout pregnancy, except over the last few days before parturition (days 20-22) when it appears that the treated groups did not gain weight when compared with the controls. This effect was only statistically significant for the 1000 mg/kg/day treatment group 21 and 22 days after mating.
Any differences in body weights seen during the latter stages of pregnancy were not reflected in the mean body weights of the ATBC treated rats during the post-partum phase of the F, generation, when there were no marked differences when compared to control body weights.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F0 generation - Males
The mean food intakes of the groups fed 300 or 1000 mg ATBC/kg/day were statistically signifkantly lower than controls over the first day of treatment. This is likely to be due to palatability of the diet at these dose levels. Over the subsequent few days the mean food intakes of these groups were higher than controls, and this difference was statistically significant in the 300 mg ATBC/kg/day group. The food intakes for all three treatment groups were generally slightly lower than control food intakes from day 21 onwards. This difference was statistically significant between days 56-63 only.
F0 generation - Females
During the pre-mating phase, the mean food intake of the group fed 1000 mg ATBC/kg/day was statistically sigzllficantly lower than controls over the first day of treatment and again this is probably due to palatability because this effect was not observed again during this phase of the study. There were other occasional differences in all treatment groups, but the food intakes were generally statistically sigmficantly higher than controls.
During the first 15 days of pregnancy, the only differences seen were slight but statistically significantly higher food intakes for the mid dose group between days 1 and 2 of pregnancy and between days 9-10 in the top dose group. From day 15 to parturition, there were numerous differences, and although these were both higher and lower than control values, the general trend was towards lower food intakes in the mid- and top-dose level groups.
There were no marked differences from control values in food intakes during the lactation phase except on one occasion, between days 14-17 postpartum where the food intake of the low dose group was statistically significantly lower than controls.

TEST MATERIAL INTAKE
F0 generation
The mean intakes for the F0 generation rats for the males and for the females during the pre-mating, pregnancy, and post-partum phases are as -
Control - 0.0 mg/kg/day (both males and females)
100 mg/kg/day - 108.8 (males), 108.8 (females)
300 mg/kg/day - 316.2 (males), 363.0 (females)
1000 mg/kg/day - 1069.1 (males), 1119.1 (females)

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

F0 generation - Males
The mean water intakes of all three groups treated with ATBC were statistically significantly lower than controls over the first day of treatment. There were other sporadic incidences of lower water intakes in the treated groups up to 21 days after the start of the study. From day 21 to 77, the mean water intakes of the group fed 1000 mg/kg/day ATBC were consistently and statistically significantly lower (80-90% of controls). Over the period of measurement from day 70 to 77, the intakes of the other two groups were also statistically significantly lower.
F0 generation - Females
During the pre-mating phase, the water intakes of the 300 and 1000 mg/kg/day ATBC groups were occasionally statistically significantly lower than controls. The water intakes of the low dose group were unaffected by treatment..
During pregnancy, the water intakes of the 100 and 300 mg/kg/day ATBC treated groups were unaffected by treatment. Contrary to this, the water intakes of the top dose group were consistently lower over the whole period of pregnancy, and these differences were statistically significantly lower at all times except between days 8-9, 11-12, 14-16, 18-19 and 20-21.
During the lactation phase, the water intakes of the top dose rats were statistically sigmficantly lower than controls between days 4-10 and 14-1 post-partum. Water intakes of the 100 and 300 mg/kg/day ATBC treated groups were unaffected during this phase of the study.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the F0 male rats, preputial abscesses, some of which had ulcerated, were seen in several animals from the control and treated groups. The incidence of this finding was 3/30, 3/30, 5/30 and 5/30 for the control, 100, 300 and 1000 mg/kg/day ATBC treated groups respectively. This lesion was also found in two F1 male rats from the top dose group.
Pyelonephritis of the kidney was seen in two F1 female pups, one each from the control and top dose groups. The lesion seen in the pup (from dam No. 306) from the 1000 mg/kg/day group was accompanied by transitional cell hyperplasia of the bladder. Nephroblastoma were present in two animals, one F0 female from the 100 mg/kg ATBC treated group, and one F1 male treated rat from the top dose group (No. 519, also from dam No. 306).
Benign mammary adenomas were present in two F1 female rats, one from each of the low and mid-dose groups, taken for necropsy on day 20/21 of pregnancy. One young F1 male rat (No. 443) from the low-dose ATBC treatment group, taken for early necropsy at the F1 datum point, was found to have a malignant anaplastic carcinoma on the lefi hind leg. This was considered to be epithelial in origin and unrelated to treatment with ATBC.
Findings in lymph nodes were seen in two rats. One of these was draining the preputial abscess seen in F1 male rat No. 509, and the other was a cystic lymph node in an F0 female (No. 298) from the top dose group.
In the liver, various different lesions were observed in the control, low and mid-dose ATBC treated groups. Although accessory lobes are common in rats (F1 female pup from control dam No. 203), the aetiology of the multiple liver abscesses (F0 male No.32 from the low dose group), the infarcted calcified degenerative lobe (F1 female pup from control dam No. 212) or the foreign body granuloma (F1 male pup from mid-dose dam No. 276) are uncertain, but are unlikely to be a result of treatment.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating and fertility
F0 generation
The time to coition and the period of gestation appeared to be unaffected in the groups of rats treated with ATBC, when compared to the controls. Although there was a slightly lower incidence in the number of F0 dams that gave birth to one or more live pups in the ATBC treated groups, no obvious dose relationship was apparent. The statistical analysis of the length of the gestation period indicates a difference from control in the 100 and 1000 mg/kg/day ATBC groups, even though the mean figures for all these groups are identical - 21.7 days. This is due to the nature of the statistical analysis method used and this effect was not considered to be relevant to this study as the mean falls within the normal limits of 21 + 1 day for the gestation period.
F1 generation
The time to coition, the period of gestation and the number of F, dams that gave birth to one or more live pups appeared to be unaffected in the groups of rats treated with ATBC, when compared to the controls. Although the statistical analysis of the length of the gestation period for the mid and high-dose rats was statistically significantly lower than that of the control group, the difference was very small and the mean length of gestation was considered to be within the normal limits. This statistical difference is probably due to the nature of the statistical analysis method used and this effect was not considered to be treatment related.

Litter weights and survival
F1 generation
The mean number of F1 generation pups per litter, produced as a result of mating F0 rats was unaffected by treatment with ATBC at all dose levels. The mortality levels of the pups produced from the groups fed ATBC at levels of 100 and 1000 mg/kg/day were slightly (mean mortality difference of 0.2 pups/litter), but statistically significantly higher than the control mortality seen between 1 and 4 days after parturition. In contrast to this, the mortality level between days 7 and 21 after parturition was slightly (mean mortality difference of 0.1 pups/ litter), but statistically si@icantly lower than the control mortality level. The differences seen in mortality levels between the control and ATBC fed groups were very small and were not considered to be related to treatment. The litter weights and mean pup weights at the highest dose were statistically signrficantly lower than controls on the day after birth (day 1) and again on day 4. Although mean
pup weight for this group was not statistically signicantly affected by treatment 7 and 14 days after birth, it was statistically sigmficantly lower than controls again on day 21. There were no marked differences in the mean ratio of male and female pups produced at each treatment level.
F2 generation
The mean number of F2 generation pups per litter, produced as a result of mating F1 rats was unaffected by treatment with ATBC at all dose levels. Although the mean litter weights were unaffected by treatment with ATBC, the mean pup weights from the high dose ATBC fed group were statistically significantly lower than those pups from the control group 1, 4, 14 and 21 days after parturition. The mean pup weight from the 300 mg/kg/day ATBC group was also statistically si~icantlylo wer than controls 21 days after parturition. Although there were no differences in pup mortality over the first 4 days after birth, there was a statistically significantly higher mortality in the top dose ATBC treated pups over the period 7-21 days after birth when compared to control mortality. The statistical analysis of the mortality over this period also indicated that the low dose pup mortality was significantly different from the controls even though the figure for this is the same as for the control group. This is due to the nature of the statistical analysis method used and this result has no toxicological significance.

as described above

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

water consumption and compound intake

reproductive performance

Remarks on result:

other:

Remarks:

reproductive toxicity

Neuropathological findings:

not examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Observations of the condition and behaviour of the F1 generation rats during their time on the study were as -

In the F1 generation male rats there was a 1/30 incidence of red staining around the eyes in the control and 300 mg/kg/day groups. One rat, number 443, from the 100 mg/kg/day ATBC treatment group, was found to have an ulcerated lump on it's left hind limb at the F1 datum point. This rat was removed for early necropsy on day 0. Lumps were also observed on the limbs of two F1 female rats, one each from the 100 and 300 mg/kg/day groups.
At necropsy of the F1 male pups, one pup from the 100 mg/kg/day group was observed to have dark staining around its right eye.
At necropsy of the F1 adult male rats, further observations of dark staining around the eyes were noted in animals from the low (2/29) and mid-dose (1/30) groups that were not previously recorded in the daily observations.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation - Males
There were no obvious differences in the mean body weights of the male rats selected as part of the F1 treatment phase up to the F1 datum point (Study day 0 for the F1 generation).
In contrast to this, the mean body weights of the groups fed ATBC at levels of 300 or 1000 mg/kg/day were consistently and statistically significantly lower than controls from the F1 datum point up to their scheduled necropsy. The mean body weights of these groups were approximately 90-95% of control values. The body weights of male rats fed ATBC at a level of 100 mg/kg/day were unaffected by treatment.
F1 generation - Females
There were no marked differences in the mean body weights of the female rats selected as part of the F1 treatment phase up to the F1 datum point (Study day 0 for the F1 generation).
During the pregnancy and post-partum phases, there were no marked differences seen in the mean body weights of rats in any of the ATBC treated groups when compared to the controls except over the last few days before parturition (days 20-21) when it appears that the treated groups did not gain weight at the same rate as the controls.
Individual body weights of the non-pregnant F1 rats were not analyzed statistically due to the small group sizes

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation - Males
There were no obvious differences in the food intakes of the male rats selected for the F1 treatment phase, in the period between weaning and the F1 datum point. Reliable analysis of data was not possible during this period due to different group sizes over each food intake measurement period.
The food intakes of the F1 male rats were, in general, unaffected by treatment with ATBC. Although there were numerous occasions when the food intakes of all three treatment groups were statistically significantly different from control values, these differences were both lower and higher than the controls and followed no pattern of dose response. As such, these differences were not considered to be treatment related.
F1 generation - Females
There were no obvious differences in the food intakes of the female rats selected for the F1 treatment phase, in the period between weaning and the F1 datum point. As for the males, reliable analysis of data was not possible during this period due to different group sizes over each food intake measurement period.
During the period between the F1 datum point and mating, statistically significant differences that were both lower and higher than controls were occasionally seen. As there was no obvious pattern to any of these changes they were not considered to be treatment related.
The food intakes of the top dose pregnant rats were generally slightly higher than the control food intakes and this difference was statistically significant between days 0-2,4-5,14-15 and 17-18 of pregnancy. There were also differences in the food intakes of the low and mid-dose groups over this period, however they did not exhibit any obvious dose-response relationship.
Individual food intakes of the non-pregnant F1 rats were not analyzed statistically due to small group sizes.
There were no differences between the food intakes of the ATBC treated groups and the control group during the post-partum phase, except on one occasion, between days 4 and 7 post-partum, where the mean food intake for the group fed ATBC at a level of 100 mg/kg/day was statistically significantly lower than that of the control group. This difference was not considered to be treatment related.

TEST MATERIAL INTAKE
F1 generation
The mean intakes for the F1 generation rats for the males and for the females during the pre-mating, pregnancy, and post-partum phases are as -
Control - 0.0 mg/kg/day (both males and females)
100 mg/kg/day - 928.8 (males), 115.4 (females)
300 mg/kg/day - 291.5 (males), 321.1 (females)
1000 mg/kg/day - 985.8 (males), 1113.5 (females)

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

F1 generation - Males
There were no obvious differences in the water intakes of the male rats selected for the F1 treatment phase, in the period between weaning and the F1 datum point, except where statistically siflicantly higher water intakes were recorded between 35 and 42 days after birth in the 300 mg/kg/day group and between days 28-35 and 42-45 after birth in the low-dose ATBC fed group. However, in general, reliable analysis of data was not possible during this period due to different group sizes over each water intake measurement period. Between 28-35 days after the F1 datum point, the water intakes of the top dose group were approximately 90% of the control group values. This difference was statistically significantly lower at all subsequent water intake measurements up to mating (day 70), and again, after mating, from day 84 to day 112, prior to necropsy. The water intakes of the low and mid-dose groups were largely unaffected by ATBC, except between days 105-112 after the F, datum point where the intakes of the 300 mg/kg/day group were slightly, but statistically significantly lower than the controls.
F1 generation - Females
The mean water intakes for the top dose group were statistically significantly lower than control water intakes between 35 and 42, and 42 and 46 days after birth. No other differences were seen in the F1 female rats selected for the F1 treatment phase between weaning and the F1 datum point. In general, reliable analysis of data was not possible during this period due to different group sizes over each water intake measurement period.
During the treatment phase between the F1 datum point and mating, water intakes that were statistically siflicantly lower than those of the control group were frequently observed in the group fed ATBC at a level of 1000 mg/kg/day. Other occasional differences noted in the low and mid dose groups were not considered to be treatment related.
During pregnancy , the water intakes of the low dose ATBC treated group were largely unaffected when compared with the controls. There were two occasions, between days 5-6 and 7-8 when the water intakes of the group fed ATBC at a level of 300 mg/kg/day were statistically sigmficantly lower than controls. The differences seen in this group were not consistent as on some occasions slightly higher water intakes were recorded. The water intakes for the top dose ATBC fed group were however consistently between 75 and 97% of the control water intakes, and this difference was statistically signuficant over the majority of the gestation period. After parturition, the differences seen in the 1000 mg/kg/day ATBC treated group during pregnancy were no longer apparent, except between days 10-14 postpartum when the water intake for this group was approximately 90% of the controls. This difference was statistically significant. Between days 17-21 post-partum, the water intake of the low dose group was statistically significantly higher than that of the controls. This finding was
not considered to be related to treatment.
Individual water intakes of the non-pregnant F1 rats were not analyzed statidtically due to small group sizes.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups
The only abnormality observed in the male F, generation pups, at either the full or gross necropsy examinations was a firm yellow mass seen at the bifurcation of the median lobe of the liver of one pup from the 300 mg/kg/day group.
In the female pups, abnormalities were observed in three control rats. These were small white foci on each kidney (from Dam No. 209), a pale raised area on the median lobe of the liver (from Dam No. 203) and a firm yellow nodule replacing one of the median lobes of a pup from Dam No. 212. No abnormalities were observed in any pups from the 100 and 300 mg/kg/day groups. In one pup from the top dose group (from Dam No. 306),the kidneys were pale, enlarged, flaccid and hollow, the bladder wall was thickened and the bladder contained a calcium deposit.

F1 generation adults
There were no abnormal macroscopic findings in any male or female control group animals. In the group fed ATBC at a level of 100 mg/kg/day, one male rat, No 443, had a 3x3 cm ulcerated mass on its left hind limb, and one female, No. 658 had five well defined subcutaneous masses. Both of these rats were taken for early necropsy. In the female group fed ATBC at a level of 300 mg/kg/day, one rat, No. 686 had a 2x3 cm lobulated, encapsulated, haemorrhagic and cystic mass. No abnormalities were seen in the male group fed ATBC at this level.
In the male group fed ATBC at a level of 1000 mg/kg/day, macroscopic lesions were observed in three rats. In rat Nos. 495 and 509, firm subcutaneous lumps were present. These were cyst-like structures filled with yellow-green coloured contents. The mesenteric lymph nodes were also attached to the dorsal aorta in this second rat. The other lesion seen, in rat No. 519 was a multi-lobulated mass on the apex of the right kidney. The cut surface presented a solid white mass extending throughout the apex of the kidney. No abnormalities were seen in any female rats treated with ATBC at this dose level.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the F0 male rats, preputial abscesses, some of which had ulcerated, were seen in several animals from the control and treated groups. The incidence of this finding was 3/30, 3/30, 5/30 and 5/30 for the control, 100, 300 and 1000 mg/kg/day ATBC treated groups respectively. This lesion was also found in two F1 male rats from the top dose group.
Pyelonephritis of the kidney was seen in two F1 female pups, one each from the control and top dose groups. The lesion seen in the pup (from dam No. 306) from the 1000 mg/kg/day group was accompanied by transitional cell hyperplasia of the bladder. Nephroblastoma were present in two animals, one F0 female from the 100 mg/kg ATBC treated group, and one F1 male treated rat from the top dose group (No. 519, also from dam No. 306).
Benign mammary adenomas were present in two F1 female rats, one from each of the low and mid-dose groups, taken for necropsy on day 20/21 of pregnancy. One young F1 male rat (No. 443) from the low-dose ATBC treatment group, taken for early necropsy at the F1 datum point, was found to have a malignant anaplastic carcinoma on the lefi hind leg. This was considered to be epithelial in origin and unrelated to treatment with ATBC.
Findings in lymph nodes were seen in two rats. One of these was draining the preputial abscess seen in F1 male rat No. 509, and the other was a cystic lymph node in an F0 female (No. 298) from the top dose group.
In the liver, various different lesions were observed in the control, low and mid-dose ATBC treated groups. Although accessory lobes are common in rats (F1 female pup from control dam No. 203), the aetiology of the multiple liver abscesses (F0 male No.32 from the low dose group), the infarcted calcified degenerative lobe (F1 female pup from control dam No. 212) or the foreign body granuloma (F1 male pup from mid-dose dam No. 276) are uncertain, but are unlikely to be a result of treatment.

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

as described above

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

< 1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

water consumption and compound intake

Remarks on result:

other:

Remarks:

reproductive toxicity

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups
No macroscopic abnormalities were observed during either the full or gross necropsy examinations of the F2 generation pups.

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The mean number of F2 generation pups per litter, produced as a result of mating F1 rats was unaffected by treatment with ATBC at all dose levels. Although the mean litter weights were unaffec ted by treatment with ATBC, the mean pup weights from the high dose ATBC fed group were statistically significantly lower than those pups from the control group 1, 4, 14 and 21 days after parturition. The mean pup weight from the 300 mg/kg/day ATBC group was also statistically signiicantly lo wer than controls 21 days after parturition. Although there were no differences in pup mortality over the first 4 days after birth, there was a statistically significantly higher mortality in the top dose ATBC treated pups over the period 7-21 days after birth when compared to control mortality. The statistical analysis of the mortality over this period also indicated that the low dose pup mortality was significantly different from the controls even though the figure for this is the same as for the control group. This is due to the nature of the statistical analysis method used and this result has no toxicological significance.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

food consumption and compound intake

Key result

Reproductive effects observed:

no

None

Conclusions:

In summary, apart from the lower water intakes of the 300 and 1000 mg/kg body weight/day ATBC treated F0 and F1 generation female rats during pregnancy, and the lower body weights of the F1 male groups fed ATBC at levels of 300 and 1000 mg/kg/day (not regarded as toxicological relevant as reduction was only between 5 to 10%), acetyl tributyl citrate did not cause any reproductive toxicity effects in the rat under the conditions of this two generation study.

Executive summary:

Four groups of 30 male and 30 female Sprague Dawley strain rats were given acetyl tributyl citrate (ATBC) in the diet at dose levels of 0 (control), 100, 300 or 1000 mg/kg/day, continuously for the time they remained part of the study. These were designated F0animals and were fed the diets containing the test article at concentrations calculated from preliminary or historical data, or from estimated food consumptions and body weights. Prior to mating, the males were treated for 77 days, and females for 21 days, when one male was paired with one female from the same treatment group for up to two weeks. After confirmation of mating, the male was removed from the female and fed the relevant diet for a further 3-8 weeks, depending upon the time of mating and scheduled necropsy.

The F0females were allowed to litter, and these animals were designated the F1generation. The litters were observed and weighed on days 1, 4, 7, 14 and 21 after birth. On day 4 after birth, each litter was culled to a maximum of 10 pups. After weaning, at least one male and one female from each litter, where possible, were randomly selected to give F1group sizes of 30 rats of each sex. These rats were fed diet containing ATBC at the relevant dose levels for up to a further 12-14 weeks and then randomly paired, avoiding sibling matings, to produce the F2generation. Four days after birth, these litters were also culled to a maximum of 10 pups.

A necropsy examination was carried out, and a full range of tissues retained from the F0and F1males and females that did not produce litters, within 42 days from their last day of pairing, and the F0and F1males and females that did produce litters, within 14 days of weaning the litter. One animal of each sex from both the F0 and F1 litters was randomly selected and also subjected to a necropsy procedure. The remaining pups from each litter were killed and the tissues examined, but only tissues that were thought to show pathological abnormalities were retained. Adult rats were fasted overnight prior to necropsy, but had free access to water. Body weights were recorded at least once weekly for all animals as long as they remained on the study. F1and F2generation female rats were weighed daily from the day that conclusive evidence of mating had occurred to the day of littering. Total litter weights (F0and F1 generations) were recorded on the day after birth, day 1, and on days 4,7 and 14. On day 21, the young were weighed individually.

The food and water intake for each cage of rats was measured between the intervals of body weight measurements, except when the F0and F1generation males and females were housed together during mating.

Each animal was observed daily for any change in its condition or behavior, and once weekly a more detailed examination was carried out. Litters were examined on the day after birth (day 1) for the numbers of alive, dead or abnormal young, on days 4,7,10,14 and 17 for the number of survivors and any abnormalities, and on day 21 for the number of survivors, sex of each pup and any abnormalities.

Cutaneous and subcutaneous lumps were a fairly common occurrence in the genital/inguinal region in the F0males of all treatment groups, including the controls, and in two of the F1males in the top dose group. When examined microscopically, these were found to be perpetual abscesses, a finding which is not uncommon in mature, sexually active male rats, and the total incidence within the study does not suggest any association with treatment.

Subcutaneous lumps, diagnosed as benign mammary adenomas were also noted in two female F1ATBC treated rats (one each from the low and mid dose groups) during the last few days of pregnancy. Apart from these observations and the occasional incidence of red staining around the eyes in the F0ATBC fed male rats, the condition and behavior of the rats used in this study was unaffected by treatment with ATBC at all dose levels.

The body weights of the F0generation rats were largely unaffected by treatment with ATBC. The body weights of the F1generation male rats fed ATBC at levels of 300 or 1000 mg/kg/day were consistently lower than controls in a pattern that appeared to be dose-related. This difference was not observed in the F, females.

Food intakes of the groups of F0and F1generation rats given ATBC in the diet were largely unaffected by treatment. The differences seen during the study did not follow any obvious dose-relationship and were not considered to be treatment related.

The water intakes of the low and mid-dose group rats were also largely unaffected by treatment. The water intakes of the male and female rats fed ATBC at a level of 1000 mg/kg/day were however, consistently lower than the controls in both generations over the majority of the study.

Mating, gestation and fertility of the F0and F1generations were unaffected by treatment with ATBC at all dose levels, as was the numbers of pups produced. The weights of the pups from the high dose rats were, in general, slightly lower than those of the controls, and this is linked with a slightly higher mortality. It is considered that these marginal effects are a consequence of reduced body weight gain and water intakes in the dams rather than any direct effect of ATBC.

At the necropsy examination, there was a low incidence of various abnormalities throughout the control and treatment groups, of each generation, none of which could be considered to be related to treatment with ATBC.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Good quality.
Due to technical reasons only a concret number can be given but actually the NOAEL is >1000, e.g. no effects were observed up to the limit dose.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on fertility via oral route:
Data from 2 -Generation study in rats were used. In addition, the toxicokinetics of ATBC were investigated in rats with oral exposure (see section 7.1.1) and the results are not indicating a bioaccumulation potential. Longer-term studies with oral dosing gave no indication for adverse effects on reproductive organs. Therefore, adverse effects concerning toxicity to reproduction are not to be expected and there is no scientific justification for planning further animal tests to investigate this endpoint.

Justification for selection of Effect on fertility via inhalation route:
Inhalation is not relevant route of exposure. No further studies are needed.

Justification for selection of Effect on fertility via dermal route:
No further studies are needed.

Effects on developmental toxicity

Description of key information

A weight of evidence approach was applied based on experimental data (in part with limited validity), QSAR predictions as well as information from oral exposure (including also in utero) in rats. The detailed information with further justification and additioanl information is given in the document provided under WoE: overall assessment.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: documentation sufficient for WoE assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of animals were provided feed containing a milk solution of the test substance (ATBC) at doses of 50 and 250 mg/kg for 12 months

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Diet (e.g. ad libitum): ad libitum
Water (e.g. ad libitum): ad libitum

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

Groups of animals were provided feed containing a milk solution of the test substance (ATBC) at doses of 50 and 250 mg/kg for 12 months

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

In the 9th month of the study, a cross-mating of the animals was performed, male gonads were evaluated and embryotoxic effects were examined. The following indicators of embryotoxic effects were evaluated:
early and late embryonic death (determined by examining the numbers of corpora lutea and implantation sites)
number of normal, resorptive and deformed tissues

Duration of treatment / exposure:

12 months

Frequency of treatment:

continuously via diet

Duration of test:

12 months

No. of animals per sex per dose:

1 replicate per dose, rest not reported

Control animals:

yes, concurrent no treatment

Maternal examinations:

Clinical signs, mortality, size and weight of the placenta

Fetal examinations:

early and late embryonic deaths (examining the numbers of corpora lutea and implantation sites)
number of normal, resorptive and deformed tissues
length of newborns
ear and eye openings, appearance of body hair and teeth, behaviour; and body weight

Statistics:

Not reported

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
50 mg/kg: no adverse effects
250 mg/kg: increases in body weight, length of the progeny and placental weight
No differences concerning fertility rate and number of pups born/pregnant female.
There were no significant effects on male gonads and at 250 mg/kg the spermatogenesis index was comparable with controls.

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No adverse effects on eye and ear opening, body fur and incisor appearance.
No effects on behaviour and body weights.

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Remarks on result:

other: No effects up to the highest dose tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Dosing with ATBC via diet over 12 months had no effects to male sexual cells, caused no embryotoxic effects and had no impact on the development in offspring.

Executive summary:

Groups of rats were dosed with a milk solution of ATBC via diet at nominal doses of 50 and 250 mg/kg over 12 months. A cross-mating of the animals was performed.

In the 9th month of the study, gonads were evaluated and the animals were evaluated for embryotoxic effects. The dosing caused no significant effects on male sexual cells, no embryotoxic effects and there also were no adverse effects on growth and foetal/litter development in the offspring.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: documentation sufficient for WoE assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of animals were provided feed containing a milk solution of the test substance (ATBC) at doses of 50 and 250 mg/kg for 12 months

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Diet (e.g. ad libitum): ad libitum
Water (e.g. ad libitum): ad libitum

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

Groups of animals were provided feed containing a milk solution of the test substance (ATBC) at doses of 50 and 250 mg/kg for 12 months

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

In the 9th month of the study, a cross-mating of the animals was performed, male gonads were evaluated and embryotoxic effects were examined. The following indicators of embryotoxic effects were evaluated:
early and late embryonic death (determined by examining the numbers of corpora lutea and implantation sites)
number of normal, resorptive and deformed tissues

Duration of treatment / exposure:

12 months

Frequency of treatment:

continuously via diet

Duration of test:

12 months

No. of animals per sex per dose:

1 replicate per dose, rest not reported

Control animals:

yes, concurrent no treatment

Maternal examinations:

Clinical signs, mortality, size and weight of the placenta

Fetal examinations:

early and late embryonic deaths (examining the numbers of corpora lutea and implantation sites)
number of normal, resorptive and deformed tissues
length of newborns
ear and eye openings, appearance of body hair and teeth, behaviour; and body weight

Statistics:

Not reported

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
50 mg/kg: no adverse effects
250 mg/kg: increases in body weight, length of the progeny and placental weight
No differences concerning fertility rate and number of pups born/pregnant female.
There were no significant effects on male gonads and at 250 mg/kg the spermatogenesis index was comparable with controls.

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No adverse effects on eye and ear opening, body fur and incisor appearance.
No effects on behaviour and body weights.

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Remarks on result:

other: No effects up to the highest dose tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Dosing with ATBC via diet over 12 months had no effects to male sexual cells, caused no embryotoxic effects and had no impact on the development in offspring.

Executive summary:

Groups of mice were dosed with a milk solution of ATBC via diet at nominal doses of 50 and 250 mg/kg over 12 months. A cross-mating of the animals was performed.

In the 9th month of the study, gonads were evaluated and the animals were evaluated for embryotoxic effects. The dosing caused no significant effects on male sexual cells, no embryotoxic effects and there also were no adverse effects on growth and foetal/litter development in the offspring.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, that is used within a weight of evidence approach based on publicly available data

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

Modified guideline study (OECD 408 with preceeding in utero exposure)

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Version / remarks:

Modified guideline study (OECD 408 with preceeding in utero exposure)

Qualifier:

according to guideline

Guideline:

other: EC Method B26

Version / remarks:

Modified guideline study (OECD 408 with preceeding in utero exposure)

Deviations:

not specified

Principles of method if other than guideline:

Modified guideline study (OECD 408 with preceeding in utero exposure)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Diet: ad lib
Water: ad lib

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

After 4 weeks of treatment, the F0 males and females were cohabitated and mated.
Males were sacrificed and toxicity was assessed as for a 28-day repeated dose study.
Females (P) were placed in separate cages and allowed to deliver, nurse and wean offspring

Duration of treatment / exposure:

F0 males and females were treated for four weeks prior to mating until scheduled sacrifice.
The F1 male and female offspring were exposed in utero and from birth until the start of the 13-wk study.
The F1 offspring selected for the 13-wk study were then provided the respective treated diets for 13-wk.

Frequency of treatment:

Continually via diet

Duration of test:

13 wk plus in utero, nursing and weaning.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

nominal in diet

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

nominal in diet

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

nominal in diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

nominal in diet

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

yes, plain diet

Details on study design:

F0 animals (25 rats/sex/group) were dosed via diet at target doses of 0, 100, 300 and 1000 mg/kg/d f or 4 weeks before pairing and throughout mating.
F0 males were sacrificed after mating and F0 females were dosed throughout gestation, littering and lactation (killed after weaning on lactation day 21).
Parentals were checked for reproductive endpoints. The F1 generation was exposed in utero and from birth until the start of the 13-week study (age of ca. 4 weeks) and then dosed (20/sex/group) as parentals. Additional 10 F1 males and females from controls and high dose group were user for a 4 week recovery period.

Maternal examinations:

Parental animals were evaluated for reproductive endpoints (mating performance, fertility, gestation length and parturition, litter size, numbers of implantations, survival and growth).
F1 animals were evaluated for sexual maturation (balano-preputial separation, vaginal opening, anogenital distance, retained areolae in males, sperm assessments), estrous cyclicity, physical appearance, ophthalmologic effects, neurobehavioral effects, growth, food consumption, survival, hematology, blood chemistry, urinalysis, peroxisome proliferation, organ weights, gross pathology and histopathology.
A full range of tissues were retained for the F0 males and females and reproductive organ tissues were retained for F1 males and females.
Microscopic examinations were performed on a standard set of tissues for F0 males and females, as well as tissues found to be abnormal at necropsy.

Ovaries and uterine content:

Animals allowed to deliver litters naturally.

Fetal examinations:

F1 animals were evaluated for sexual maturation (balano-preputial separation, vaginal opening, anogenital distance, retained areolae in males, sperm assessments), estrous cyclicity, physical appearance, ophthalmologic effects, neurobehavioral effects, growth, food consumption, survival, hematology, blood chemistry, urinalysis, peroxisome proliferation, organ weights, gross pathology and histopathology.
A full range of tissues were retained for the F0 males and females and reproductive organ tissues were retained for F1 males and females.

Statistics:

For organ weights and body weight changes, homogeneity of variance was tested using Bartlett’s test followed by Behrens-fisher test or Dunnett’s test as appropriate.
Macroscopic pathology and histopathology data were assessed using Fisher’s Exact test. Estrus cycles were analyzed using the Cochran-Armitage trend test.
Other statistical tests used as appropriate were: Williams’ test for a dose-related response; Student’s t-test; Shirley’s non-parametric test for a dose-related response; Steel’s test; and Wilcoxon rank sum test.
Significance level was p<0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Details on maternal toxic effects:

Estrous cycles, mating performance, fertility, gestation length and parturition, were all unaffected by treatment. Litter size, survival and growth were similar in all groups and within expected historical control ranges. Although numbers of implantations and litter size at 1000 mg/kg/day were marginally lower than concurrent control group levels, they were within the laboratory’s historical control ranges.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: weak peroxisome proliferation only in males

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: weak peroxisome proliferation only in females

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: numbers of implantations and litter size, but within historical control range

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Anogenital distance and sexual maturation in both sexes and retention of areolae in male offspring were unaffected by treatment. There were no adverse effects on sperm motility, counts or morphology.
There were no findings at necropsy of surplus offspring that were considered to be treatment-related.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Estrous cycles, mating performance, fertility, gestation length and parturition, were all unaffected by treatment. Litter size, survival and growth were similar in all groups and within expected historical control ranges. Although numbers of implantations and litter size at 1000 mg/kg bw/day were marginally lower than concurrent control group levels, they were within the laboratory’s historical control ranges. Anogenital distance and sexual maturation in both sexes and retention of areolae in male offspring were unaffected by treatment. There were no adverse effects on sperm motility, counts or morphology. There were no findings at necropsy of parental animals or surplus offspring that were considered to be treatment-related. Parental and offspring NOAELs were 300 and 1000 mg/kg bw/day for reproductive and developmental endpoints, respectively.

Conclusions:

The NOAEL values for systemic toxicity were given with 100 mg/kg/d for males and with 300 mg/kg/d for females.
NOAEL values for reproduction and developmental toxicity: 300 mg/kg/d for parental animals and 1000 mg/kg/d for offspring.

Executive summary:

In this study F0 males and females were treated for four weeks prior to mating until scheduled sacrifice. The F1 male and female offspring were exposed in utero and from birth until the start of the 13-week study. The F1 offspring selected for the 13-week study were then provided the respective treated diets for 13-weeks. The target doses were 0, 100, 300 and 1000 mg/kg/d. Treatment at 1000 mg/kg/d resulted in a slight reduction in body weight gain in both sexes and liver weights were increased and hepatic hypertrophy occurred at 1000 mg/kg/d in both sexes. There was a weak peroxisome proliferation in males at >= 300 mg/kg/d and in females at 1000 mg/kg/d. The NOAEL values for systemic toxicity were given with 100 mg/kg/d for males and with 300 mg/kg/d for females.

Reference 4

Endpoint:

developmental toxicity

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Version / remarks:

Danish QSAR Database (DTU)

Specific details on test material used for the study:

smiles C(=O)(C(CC(=O)OCCCC)(CC(=O)OCCCC)OC(C)=O)OCCCC

Species:

other: not applicable for QSAR

Remarks on result:

other: QSAR prediction, see Text

Remarks on result:

other: QSAR prediction, see Text

Key result

Developmental effects observed:

no

The three Models for Endocrine endpoints and model for Teratogenic Potential in Humans of the Danish QSAR Database (DTU) predicts that ATBC is negative. The substance is inside the applicability domain.

Conclusions:

The results from three QSAR models in the Danish QSAR Database (DTU) predicts that ATBC is negative. The substance is inside the applicability domain

Reference 5

Endpoint:

developmental toxicity

Type of information:

other: Overall assessment/weight of evidence statement

Adequacy of study:

other information

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Good quality.
Due to technical reasons only a concret number can be given but actually the NOAEL is >1000, e.g. no effects were observed up to the limit dose. Thefore, there is also no need for an aditioanl safety factor due to the missing information from the second species.

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

Justification for selection of Effect on developmental toxicity: via oral route:
Weight of evidence approach was applied using information from literature data, repeated dose toxicity studies as well as QSAR prediction.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Inhalation is not relevant route of exposure. No further studies are needed.

Justification for selection of Effect on developmental toxicity: via dermal route:
No further studies are needed. The feeding developmental study in rats is sufficient to cover possible effects after dermal exposure.

Justification for classification or non-classification

Toxicity to reproduction was not observed at dose levels up to 1000 mg/kg/day in a two-generation reproductive toxicity study nor in a 13-week toxicity study with an in utero exposure phase supported by the weight of evidence approach. Also several repeated dose toxicity studies gave no indications for adverse effects on reproductive organs.

Therefore, there is no need for classification of ATBC.