reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Between 30 July 2009 and 12 July 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No.415 “One Generation Reproduction Toxicity Study” (adopted 26 May 1983).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): OS 162170N
- Molecular formula (if other than submission substance): C21H34O3
- Molecular weight (if other than submission substance): 334.491
- Smiles notation (if other than submission substance): Oc1c(C(C)(C)C)cc(cc1C(C)(C)C)CCC(=O)OCCCC
- InChl (if other than submission substance): 1S/C21H34O3/c1-8-9-12-24-18(22)11-10-15-13-16(20(2,3)4)19(23)17(14-15)21(5,6)7/h13-14,23H,8-12H2,1-7H3
- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: alkylated phenol
- Physical state: amber coloured solid block
- Analytical purity: approximately 98%
- Stability under test conditions: The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the sponsor.
- Storage condition of test material: room temperature in the dark
- Other:

Test animals

Species:

rat

Strain:

other: Wistar Han™:HsdRccHan™:WIST strain rat

Details on test animals or test system and environmental conditions:

Test Animals
- Source:
Wistar Han™:HsdRccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.

- Age at study initiation:
Approximately 5 to 9 weeks old

- Weight at study initiation:
154 to 225g (male); 124 to 169g (female)
- Fasting period before study:
Not applicable

- Housing:
During the pre-mating phase, animals were housed in groups of four in polypropylene cases with stainless steel mesh lids and furnished with softwood flake bedding (Datesand Ltd, Cheshire, U.K.).

During the mating phase, animals were be paired on a one male: one female basis within each dose group in polypropylene cages with stainless steel mesh lids and grid flooring, suspended over polypropylene trays lines with absorbent paper.

Following mating the males were re-housed in their original cages and the females were individually housed in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding for the gestation, birth and lactation phases of the study.

- Diet:
The animals were allowed free access to food. Rodent PMI 5002 (certified) Ground Diet (BCM IPS Limited, London, UK) was used throughout the study period. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Water:
ad libitum mains water was supplied from polycarbonate bottles attached to the cage. The water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Acclimation period:
For either 14 days (males) or 13 days (females)

ENVIRONMENTAL CONDITIONS

- Temperature:
21 ± 2 °C

- Humidity:
55 ± 15 %

- Air changes (per hr):
At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light):
12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
30 July 2009 (first day of treatment) and 05 December 2009 (final necropsy).

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

other: Basal laboratory diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Not applicable

DIET PREPARATION
-Yes (see attached Appendix 17)

The test material was incorporated into the diet at concentrations of 500, 1200 and 2500 ppm as follows: a known amount of test material was melted at 50°C and was mixed with a small amount of basal laboratory diet for ninety minutes at a constant speed, setting 1 in a Hobart QE200 mixer. This pre-mix was then added to a set amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer.

Test material formulations were analysed for concentration of OS162170N at Harlan Laboratories Ltd., Shardlow, UK Analytical Services during the study period.

The results indicate that the prepared formulations were within acceptable limits for the purpose of this study.

- Rate of preparation of diet (frequency):
The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services under project number 0525-0900. Results from the previous study showed the formulations to be stable for at least six weeks. Formulations were therefore prepared at least once a month.

- Mixing appropriate amounts with (basal laboratory diet):
See above

- Storage temperature of food:
Room temperature in the dark.

VEHICLE
Basal laboratory diet (see attached Addendum 1 for Diet Certificate of Analysis)

- Justification for use and choice of vehicle (if other than water):
Not applicable

- Concentration in vehicle:
0, 500, 1200 and 2500 parts per million (estimated achieved dosage 0, 44, 104 and 205 mg/kg bodyweight per day)

- Amount of vehicle (if gavage):
Not applicable

- Lot/batch no. (if required):
Not applicable

- Purity:
Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of OS162170N in the dietary admixtures was determined by high performance liquid chromatography (HPLC) using an external standard technique. The dietary admixtures were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 20 ppm. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 20 ppm.

The homogeneity, stability and linearity determinations were performed under Harlan Laboratories Ltd, project number 0525-0900.

The dietary admixtures were sampled and analysed within seven days of preparation

The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

See attached Appendix 17 - Chemical Analysis of Test Material Formulations, Methods and Results

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)
- Length of cohabitation: Up to twenty-one days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No
- Further matings after two unsuccessful attempts: No
- Proof of pregnancy:
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
- After successful mating each pregnant female was individually caged:
Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

The oral dietary administration of the test substance to rats for a period of up to 128 consecutive days

Frequency of treatment:

The test material was administered continuously by dietary admixture.

Duration of test:

The in-life phase of the study was conducted between 30 July 2009 (first day of treatment) and 05 December 2009 (final necropsy).

Doses / concentrations

Remarks:

Doses / Concentrations:
Dose levels of 0, 500, 1200 and 2500 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

0 ppm – control: 24 animals per sex.
500 ppm - 24 animals per sex.
1200 ppm - 24 animals per sex.
2500 ppm - 24 animals per sex.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Based on previously established toxicity data (including a 90 day dietary toxicity study - Harlan Project No. 0525-0900).

- Rationale for animal assignment (if not random):
Random

- Rationale for selecting satellite groups:
Not applicable

- Post-exposure recovery period in satellite groups:
Not applicable

- Section schedule rationale (if not random):
Random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily (see attached Table 3 and Appendices 1 and 10).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded for males on Day 1 (prior to treatment) and then weekly until termination. For females, individual bodyweights were recorded on Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Days 0, 7, 14 and 21 post coitum and on Days 1, 4, 7, 14 and 21 of lactation (see attached Tables 4 and 5, Figures 1 and 2 (Graphics) and Appendices 2 and 3).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Dietary intake for males and females was recorded weekly for each cage group until pairing. Dietary intake for males was recorded weekly following the pair in phase until termination. Following confirmation of mating, dietary intake for females was recorded on Days 0 to 7, 7 to 14 and 14 to 21 post coitum and Days to 4, 4 to 7, 7 to 14 and 14 to 21 of lactation (see attached Tables 6 and 7, Figures 3 and 4 (Graphics) and Appendices 4 and 5).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food efficiency (the percentile ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during the premating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the gestation and lactation phases of the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily by visual inspection of water bottles for any overt changes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: On completion of the dosing period all surviving males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. Females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 21 post partum.
- Organs examined: Yes (Table 2) (see also attached Table 13, 15, 16 and Appendices11-15).

In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 0.5% ammonium polysulphide solution where applicable (Salewski 1964). All animals (including offspring) were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

OTHER:

REPRODUCTION

Mating:
Surviving males were paired with females on a one male: one female basis within each dose group, for a period of up to twenty-one days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

i) Date of pairing/mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Litter Data
On completion of parturition the number of live and dead offspring was recorded. On Day 1 post partum, all surviving offspring within each litter were individually identified using ink tattoos on the feet (and tails).
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily
iii) The sex of individual offspring was recorded on Days 1, 4, 7, 14, 21 post partum
iv) Clinical condition of offspring from birth to weaning
v) Individual offspring and total litter weights on Days 1, 4, 7, 14 and 21 post partum
vi) Necropsy findings of offspring

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
Yes
Examinations included:
- Gravid uterus weight:
Yes

- Number of corpora lutea:
Yes

- Number of implantations:
Yes

- Number of early resorptions:
No

- Number of late resorptions:
No

- Other:
Parameters in Table 2 were examined in this study.

Fetal examinations:

STANDARDISATION OF LITTERS
Not applicable

PARAMETERS EXAMINED
Litter Data
For each litter the following was recorded:
i) Number of offspring born
ii) Number of live offspring will be recorded daily
iii) Sex of offspring will be recorded on Days 1, 4, 7, 14 and 21 post partum
iv) Clinical condition of offspring from birth to weaning
v) Individual offspring weights on Days 1, 4, 7, 14 and 21 post partum
vi) Necropsy findings of dead offspring and offspring killed in extremis. Particular attention will be applied to those offspring found dead at, or shortly after parturition

Statistics:

The volume of statistical references exceeds the storage capacity in this section it has therefore been included as an attachment titled 0525-0901 reproductive and offspring viability indices and statistics.

Indices:

See attachment - 0525-0901 Reproductive and offspring viability indices and statistics.

Historical control data:

Included: see Addenda 2 to 4

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Details on results

RESULTS

Adult Responses

Mortality
There were no unscheduled deaths that were considered to be related to test material toxicity.

One male treated with 500 ppm was killed in extremis on Day 57. No tissues were retained from this animal, therefore no histopathological assessment could be performed. In the absence of a dose-related response the death of this animal is considered to be of no toxicological significance.

There were no further unscheduled deaths.

Clinical Observations
See attached Table 3 and Appendix 1.

There were no toxicologically significant findings detected in the study.

Episodes of staining around the eyes were seen in one control and one 500 ppm male. Generalised scab formation was seen in one control female. Generalised fur loss was also noted in two control and 500 ppm females and one 1200 ppm female. These findings are commonly seen amongst laboratory maintained animals and are considered incidental findings of no toxicological significance.
Prior to being killed in extremis the 500 ppm male showed tiptoe gait, lethargy, hunched posture, emaciation and pallor of the extremities. In the absence of a dose-related response the findings observed in this animal are considered to be of no toxicological importance.

Bodyweight
See attached Table 4 and Table 5, Figure 1 and 2 (graphics) and Appendix 2 and Appendix 3.

During maturation, gestation and lactation a statistically significant reduction in bodyweight development was evident throughout the treated females, in comparison to controls. The reduction during this time ranged from P<0.05 to P<0.001.

No such effect was detected in the male treatment animals.

Males treated with 2500 ppm showed a statistically significant reduction (P<0.01) in bodyweight gains during Week 1 only. Males treated with 1200 ppm showed a statistically significant increase (P<0.05) in bodyweight gains during Week 14 only. In the absence of an effect on overall bodyweight gain, these isolated bodyweight changes are considered to be of no toxicological significance.

Food Consumption and Food Efficiency
See attached Table 6 and 7, Figure 3 and 4 (graphics) and Appendices 4 and 5.

A statistically significant reduction in dietary intake was evident in females treated with 1200 and 2500 ppm during Week 1 of maturation (P<0.05 and P<0.001 respectively). The reduction in food efficiency was also evident for all female treatment animals during this period with the significance ranging from P<0.01 to P<0.001. During gestation a reduction in dietary intake was evident throughout the female treatment groups, in comparison to controls. The reduction showed a dose-related response. During lactation a statistical significance for the 500 ppm females was only evident during Week 2 (P<0.05). The 1200 ppm females showed a statistical significance of P<0.05 during Weeks 2 and 3, whilst the reduction in the 2500 ppm females was seen between Weeks 1 and 3 and ranged from P<0.01 to P<0.001.

No such effect was detected in male treatment animals.

Water Consumption

There were no intergroup differences in water intake for treated animals in comparison to controls.

REPRODUCTIVE PERFORMANCE

Mating
See attached Table 2 and Table 8 and Appendix 6.

There were no intergroup differences in mating performance or gestation lengths.

Fertility
See attached Table 2 8, 9 and 10 and Table 8 and Appendices 6 to 8.

Pregnancy was achieved for all test females and twenty three control females.

One control female did show corpora lutea however, no implantation sites were evident and therefore this female was considered to be non-pregnant. Two females treated with 2500 ppm had corpora lutea and implantation sites and were therefore considered to be pregnant, but did not go on to produce a viable litter. One 500 ppm female had a total litter loss on Day 3 of lactation.

Gestation Length
See attached Table 8 and Appendix 6.

There was no adverse effect on gestation length between treated and control females.

Litter Response
No treatment related effects were detected for offspring during the daily observations.

Offspring Litter Size and Viability
See attached Tables 9 to 11 and Appendices 7 to 9.

There was no adverse effect on sex ratio or viability for treated animals in comparison to controls.

Offspring Growth and Development
See attached Tables 9 and 12 and Appendices 7 and 10.

A reduction in offspring bodyweight gain was evident throughout the lactation period in litters from all treatment groups. The reduction showed a dose-related response however, statistical significance was only achieved in the 500 ppm females during Days 7 to 14. During the lactation period the reduction in the 1200 ppm animals ranged from P<0.05 to P<0.01 whilst the 2500 ppm animals ranged from P<0.01 to P<0.001. Subsequently a statistically significant reduction in litter weights was evident throughout the lactation period for treated animals in comparison to controls (P<0.01 on Day 4 and P<0.001 on Days 7, 14 and 21 for the 2500 ppm animals: P<0.05 on Day 7 and P<0.01 on Days 14 and 21 for the 1200 ppm animals: P<0.05 for Days 4 and 21 and P<0.01 for Days 7 and 14 for the 500 ppm animals).

There was no adverse effect on offspring litter size for treated animals in comparison to controls.

The observations detected are commonly seen amongst post partum offspring and are not considered to be of any toxicological significance.

Pathology

Necropsy

See attached Tables 14 and 15 and Appendices 13 and 14.
Adults

Six males treated with 2500 ppm showed an enlarged thyroid, one of these also had small seminal vesicles. Another male from this treatment group had a small prostate and seminal vesicles, whilst a further male and two females had pale lungs with foci. In the absence of any histopathological correlates in the seminal vesicles or prostate these macroscopic findings are considered to be incidental and of no toxicological importance.

One male treated with 1200 ppm and one treated with 500 ppm had an enlarged thyroid.

No macroscopic abnormalities were detected in the remaining animals at terminal kill.

The 500 ppm male which was killed in extremis was observed with a fluid filled and ulcerated stomach.

Offspring
There were no treatment-related macroscopic abnormalities detected at terminal kill.

One 1200 ppm litter all showed patchy fur, whist one male pup from another 1200 ppm litter had a deformed tail. One 500 ppm litter had a small female pup at necropsy. Such findings often occur in studies of this nature and are considered incidental and of no toxicological importance.

Organ Weights
See attached Table 13 and Appendix 11 and Appendix 12.

A statistically significant increase in liver weights (P<0.01) both absolute and relative to terminal bodyweight was evident in animals of either sex treated with 2500 and 1200 ppm and females only treated with 500 ppm. In addition, a statistically significant increase in thyroid weights (P<0.001) both absolute and relative to terminal bodyweight was also detected throughout the female treatment animals, in comparison to controls.

No such effect was detected in males treated with 500 ppm.

A statistically significant increase both absolute and relative to terminal bodyweight was seen in the testes and pituitary weights (P<0.01 and P<0.05 respectively) for males treated with 2500 ppm in comparison to controls. The majority of the individual values are within normal range for the rats of the strain and age used and in the absence of a histopathological correlate these findings are considered to be of no toxicological significance.

A statistically significant reduction in spleen weights (P<0.001) both absolute and relative to terminal bodyweight was seen in the 2500 and 1200 ppm females. The statistically significant reduction (P<0.01) also extended to the absolute weights only of the 500 ppm females, in comparison to controls. In addition, a statistically significant reduction in uterus weights both absolute and relative to terminal bodyweight was seen in the 2500 and 1200 ppm females (P<0.001 absolute, P<0.01 relative 2500 ppm: P<0.01, 1200 ppm). Furthermore, a statistically significant reduction in absolute ovary weights (P<0.05) was seen in the 2500 and 1200 ppm females. Finally, a statistically significant reduction in absolute pituitary weights was evident throughout the female treated animals (P<0.001 1200ppm: P<0.01, 2500 and 500 ppm), in comparison to controls. The majority of the individual values are within normal range for the rats of the strain and age used and in the absence of a histopathological correlate these findings are considered to be of no toxicological importance.

Histopathology
See attached Table 16 and Appendix 15.

One control paired female was found to be non-pregnant. There were no histopathological changes in either the paired male or female animals to which failed reproductive performance could be attributed.

The following treatment-related changes were observed:

Liver: Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 2500 or 1200 ppm (P<0.001) and females only treated with 500 ppm (P<0.05).

Thyroid gland: A greater incidence and severity of follicular cell hypertrophy was seen as a consequence of treatment for animals of either sex treated with 2500 ppm (P<0.001) or 1200 ppm (P<0.01 for males and P <0.001 for females), and females only treated with 500 ppm (P<0.001).
Lung: A higher incidence of groups of alveolar macrophages was seen for males treated with 2500 ppm (P<0.01) compared with the control group. Females were not similarly affected.

Bone Marrow: A generally lower cellularity of the bone marrow, as evidenced by higher grades of severity of adipose infiltration, was observed for females treated with 2500 or 1200 ppm (P<0.001 and P<0.05 respectively). A similar effect was also seen for males at all dose levels although statistical significances were variable; statistical significance was attained at the 1200 and 500 ppm dose levels (P<0.001) but not at the 2500 ppm dose level. In the absence of a dose-related response the effect observed in the males is considered to be of no toxicological significance. The effects observed in the females are considered to be a result of functionality as there was no systemic effect or indication of change in the haematological parameters that were observed in the previously performed study, Harlan Laboratories project number 0525-0900. Therefore, the effects seen are considered to be non-adverse.

OTHER HISTOPATHOLOGY

The following conditions warrant specific mention:

Adrenal gland: No treatment-related changes were seen. Vacuolation of cortical cells is a frequently observed background condition, more especially among male rats.

Pituitary: No treatment-related changes were seen. Vacuolation of pars anterior cells is seen occasionally among male rats as a spontaneous change.

Testis/Epididymis: No treatment-related changes were seen. Testicular atrophy was seen for one control male and Leydig cell atrophy for one high dose male.
Seminal Vesicles/Coagulating gland: No treatment-related changes were seen. There was a marginally greater incidence of depletion of secretory content of one or both vesicles among high dose males but this condition is seen occasionally as a spontaneous change and may also appear as artefact.

Prostate: Prostatitis and reduced secretory content are seen occasionally as spontaneous changes.

Ovary: No treatment-related changes were seen. Ovarian cysts are seen occasionally as a spontaneous change.

Uterus: Areas of haemorrhage and fibrosis were seen in the myometrium and adjacent connective tissue of the uterus in the majority of females examined from the control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat. Dilatation of the uterine horns is a commonly observed cyclical change.

Vagina: Keratinisation of the vaginal epithelium is a normal cyclical change.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

2 500 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

2 500 ppm (nominal)

Based on:

test mat.

Remarks:

reproduction and offspring development

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined. Remark: clinical signs, viability, litter size, bodyweight, and macroscopic abnormalities were examined

Details on embryotoxic / teratogenic effects:
Embryotoxic and teratogenic effects: no examination of caesarean litter data was performed in this study. However, offspring development was assessed by the following post natal examinations:

Clinical signs
The observations detected are commonly seen amongst post partum offspring and are not considered to be of any toxicological significance

Litter Response
No treatment related effects were detected for offspring during the daily observations.

Offspring Litter Size and Viability
See attached 9 to 11 and Appendices 7 to 9.
There was no adverse effect on sex ratio or viability for treated animals in comparison to controls.

Offspring Growth and Development
See attached Tables 9 and 12 and Appendices 7 and 10.
A reduction in offspring bodyweight gain was evident throughout the lactation period in litters from all treatment groups. The reduction showed a dose-related response however, statistical significance was only achieved in the 500 ppm females during Days 7 to 14. During the lactation period the reduction in the 1200 ppm animals ranged from P<0.05 to P<0.01 whilst the 2500 ppm animals ranged from P<0.01 to P<0.001. Subsequently a statistically significant reduction in litter weights was evident throughout the lactation period for treated animals in comparison to controls (P<0.01 on Day 4 and P<0.001 on Days 7, 14 and 21 for the 2500 ppm animals: P<0.05 on Day 7 and P<0.01 on Days 14 and 21 for the 1200 ppm animals: P<0.05 for Days 4 and 21 and P<0.01 for Days 7 and 14 for the 500 ppm animals).

There was no adverse effect on offspring litter size for treated animals in comparison to controls.

The observations detected are commonly seen amongst post partum offspring and are not considered to be of any toxicological significance.

Necropsy
See attached Tables 14 and 15 and Appendices 13 and 14.

There were no treatment-related macroscopic abnormalities detected at terminal kill.

One 1200 ppm litter all showed patchy fur, whilst one male pup from another 1200 ppm litter had a deformed tail. One 500 ppm litter had a small female pup at necropsy. Such findings often occur in studies of this nature and are considered incidental and of no toxicological importance.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

See attached (0525 -0901) Tables, Figures, Appendices, Addenda and Statistics.

Applicant's summary and conclusion

Conclusions:

The administration of the test item by dietary admixture to rats for either a period of up to one hundred and twenty seven consecutive days for males or at least fifteen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for females, resulted in adult systemic effects at dose levels of up to 2500 ppm. Reductions in bodyweight development and dietary intake were observed together with increased liver and thyroid organ weight measurements. In addition, histopathological changes in the liver, thyroid, lung and bone marrow were observed. The effects detected at dietary concentrations up to 2500 ppm would not be categorised as an adverse effect of treatment according to criteria outlined in ECETOC Technical Report 85 (2002). The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 2500 ppm. There were no treatment related effects upon fertility and reproductive performance at dose levels up to 2500 ppm. There were no effects upon prenatal offspring viability and development. The reductions in offspring postnatal bodyweight development during lactation correlate with adult adaptive responses and therefore reflect a further manifestation of the same treatment effects. The change is therefore adaptive in nature. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproduction and offspring development is 2500 ppm.

Executive summary:

ORAL (DIETARY) ONE GENERATION REPRODUCTION TOXICITY STUDY IN THE RAT (OECD 415)

Introduction: The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat to assess prenatal and postnatal development, and is compatible with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).

Methods: The test material was administered by dietary admixture to three groups, each of twenty-four male and twenty-four female Wistar Han™:HsdRccHan™:WIST strain rats, at dose levels of 500, 1200 and 2500 ppm. A control group of twenty-four males and twenty-four females was treated with basal laboratory diet.

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study.

After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum.

All surviving males were terminated following the completion of a successful mating. All females and surviving unselected offspring were terminated on Day 21 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. 

Results:

Adult Responses:

Mortality: There were no unscheduled deaths that were considered to be related to test material toxicity.

Clinical Observations: There were no toxicologically significant findings detected in the study.

Bodyweight: Reductions in bodyweight development were evident during maturation, gestation and lactation for all treated females in comparison to controls. No such effect was detected in the male treatment animals.

Food Consumption and Food Efficiency: Females treated with 1200 and 2500 ppm showed a reduction in dietary intake during maturation. A reduction in food efficiency was also evident for all female treatment animals during Week 1 of maturation. During gestation and lactation a reduction in dietary intake was evident in all the female treated groups, in comparison to controls. No such effect was detected in male treatment animals.

Water Consumption: There were no intergroup differences in water intake.

Reproductive Screening:

Mating: There were no intergroup differences in mating performance.

Fertility: Pregnancy was achieved for all test animals and 23 controls. One control female did not achieve pregnancy. Two females treated with 2500 ppm had corpora lutea and implantation sites, however, failed to produce a viable litter. One female treated with 500 ppm had a total litter loss by Day 3 of lactation.

Gestation Lengths: There was no adverse effect on gestation lengths between treated and control females.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. There was no adverse effect on sex ratio or viability for treated animals in comparison to controls.

Offspring Growth and Development: A reduction in litter weights was evident throughout the lactation period for treated animals in comparison to controls. A reduction in bodyweight development was evident throughout the lactation period for treated animals of either sex in comparison to controls. There was no adverse effect on offspring litter size for treated animals in comparison to controls.

Offspring Observations: No treatment related effects were detected for offspring during the daily observations.

Pathology: Necropsy. Adults:

Six males treated with 2500 ppm showed an enlarged thyroid, one of these also had small seminal vesicles. Another male from this treatment group and two females had pale lungs with foci. One male treated with 1200 ppm and one male treated with 500 ppm had an enlarged thyroid. No macroscopic abnormalities were detected in the remaining animals at terminal kill. The 500 ppm male which was killed in extremis was observed with a fluid filled and ulcerated stomach.

Pathology: Necropsy. Offspring: There were no treatment-related macroscopic abnormalities detected at terminal kill.

Organ Weights: Males treated with 2500 and 1200 ppm showed an increase in liver and thyroid weights, in comparison to controls. An increase in liver and thyroid weights was also detected throughout the female treatment animals. No such effect was detected in males treated with 500 ppm.

Histopathology: The following treatment-related changes were observed: LIVER: Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 2500 or 1200 ppm and females only treated with 500 ppm. THYROID GLAND: A greater incidence and severity of follicular cell hypertrophy was seen as a consequence of treatment for animals of either sex treated with 2500 or 1200 ppm, and females only treated with 500 ppm. LUNG: A higher incidence of groups of alveolar macrophages was seen for males treated with 2500 ppm in comparison to controls. Females were not similarly affected.

BONE MARROW: A generally lower cellularity of the bone marrow, as evidenced by higher grades of severity of adipose infiltration, was observed for females treated with 2500 or 1200 ppm.

Conclusion: The administration of OS162170N by dietary admixture to rats for either a period of up to one hundred and twenty seven consecutive days for males or at least fifteen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for females, resulted in adult systemic effects at dose levels of up to 2500 ppm. Reductions in bodyweight development and dietary intake were observed together with increased liver and thyroid organ weight measurements. In addition, histopathological changes in the liver, thyroid, lung and bone marrow were observed. The effects detected at dietary concentrations up to 2500 ppm would not be categorised as an adverse effect of treatment according to criteria outlined in ECETOC Technical Report 85 (2002). The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 2500 ppm. There were no treatment related effects upon fertility and reproductive performance at dose levels up to 2500 ppm. There were no effects upon prenatal offspring viability and development. The reductions in offspring postnatal bodyweight development during lactation correlate with adult adaptive responses and therefore reflect a further manifestation of the same treatment effects. The change is therefore adaptive in nature. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproduction and offspring development is 2500 ppm.