Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
09 Jan 2009 - 21 Aug 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 426 (Developmental Neurotoxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, Testing Guidelines for Toxicity Studies (2-1-17), 12 Nohsan No 8147, 2000-11-24
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trade name
- Chemical name: Glycerides, castor-oil-mono-, hydrogenated, acetates
- Main component: 12-Acetoxy-octadecanoic acid (2,3-diacetoxy)propyl ester, 12-Acetoxy-octadecanoic acid 2-acetoxy-1-acetoxymethyl-ethyl ester
- Physical state: amber coloured liquid
- Analytical purity: 95% (definition of purity based on content of fully acetylated monoglycerides of major fatty acids of fully hydrogenated castor oil)
- Purity test date: 2009-04-28
- Lot/batch No.: item 175540 batch 4010534806
- Expiration date of the lot/batch: 2009-12-31
- Storage condition of test material: room temperature in the dark


Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, UK
- Age at study initiation: (P) 7-8 weeks
- Weight at study initiation: (P) 216-361 g (males) and 149-249 g (females)
- Housing: All P animals were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually (or with their litter), in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK). For the offspring selected to form the F1 generation, this housing procedure was repeated. The P and F1 offspring selected for assessment of developmental neurotoxicity were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: ground diet (Rodent PMI 5002 Diet, BCM IPS Limited, London, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each concentration of the test material was prepared by weighing an appropriate amount and mixing it with a small amount of the required volume of diet. Once this was adequately mixed the formulation was then transferred to a Hobart H800 mixer and mixed with the remaining required volume of diet.

DIET PREPARATION
- Mixing appropriate amounts with: laboratory diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the dietary admixtures was determined by gas chromatography using an external standard technique. The dietary admixtures were sampled and analysed within 2 weeks of preparation. Homogeneity and stability determinations were performed under Harlan Laboratories Ltd. project number 2384/0006. The results showed that achieved concentrations were between 95 and 121% of nominal concentration.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: 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.
- After 14 days of unsuccessful pairing replacement of first male by another male or extension of the mating phase to a third week.
- After successful mating each pregnant female was caged: individually
Duration of treatment / exposure:
(P) Males: 10 weeks during maturation and throughout mating, gestation and until completion of the P female lactation phase.
(P) Females: 10 weeks during maturation and throughout mating, gestation and until Day 21 lactation phases.
(F1) Females: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F1) Males: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F2) Males/ Females: from weaning (Day 21 of age) to termination at Day 70 of age.
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 21 of age (unselected offspring)/ Day 70 of age (selected offspring for developmental neurotoxicity)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1500, 6000 and 25000 ppm
Basis:
other: nominal in diet: animals in the high dose group initially received 15000 ppm rising to 20000 ppm to 25000 ppm during the maturation phase of each generation
Remarks:
Doses / Concentrations:
109, 435 and 1342 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 males)
Remarks:
Doses / Concentrations:
160, 630 and 2262 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 females)
No. of animals per sex per dose:
28 P males, 28 P females
24 F1 males, 24 F1 females
Control animals:
other: yes, laboratory diet treated with Arachis oil to ensure comparable calorific intake
Details on study design:
- Dose selection rationale: The dietary levels were based on known toxicology data including an earlier preliminary study in the rat (Project number 2384/0006)
- Other: In each generation, animals receiving the high dose level initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Animals were receiving the highest inclusion level by the end of the maturation/pre-pairing phase in both generations and therefore were receiving the higher dietary inclusion by the time of the main reproductive assessment.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week and once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, prior to treatment, weekly throughout the maturation phase of each generation and continuing for males until termination. Following pairing P/F1 females were weighted daily until mating was evident. Mated females were weighted on Day 0, 7, 14 and 21 post coitum and for females that littered on Day 1, 4, 7, 14 and 21 post partum. P/F1 offspring (selected for post-weaning assessment of developmental neurotoxicity) were weighted on Day 28, 35, 42, 49, 56, 63 and 70 of age. Body weight was recorded for all animals on the Day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption was assessed for each cage of adults during the maturation period and for males after mating/pairing. Females showing evidence of mating, food consumption was recorded for the periods Day 0-7, 7-14 and 14-21 post coitum. For females that littered, food consumption was recorded for the period covering Day 1-4, 4-7, 7-14 and 14-21 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: daily by visual inspection for any overt change

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: cervical, thoracic, and abdominal viscera. Adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions were weighed and/or preserved for histopathological examination.
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
Fetal examinations:
- External examinations: Yes: all unselected offspring
- Gross examination of dead pups: Yes, dead offspring was subjected to a necropsy exmination (internal and external)
Statistics:
Data were initially assessed for homogeneity of variance using Levene´s test. Where variances were shown to be homogenous, a parametric assessment of the data was performed using one way analysis of variance (ANOVA), which if significant was followed by pairwise comparisons using Dunnett´s test. Where Levene´s test showed unequal variances, the data were analysed using non-parametric methodology: Kruskal-Wallis ANOVA which, if significant, was followed by Mann-Whitney U-test. Probability values (p) are presented as follows:
p ≤ 0.001***; p ≤ 0.01**, p ≤ 0.05*, p > 0.05 (not significant).
Indices:
- Sex Ratio (% males): (Number of male offspring/ total number of offspring) x 100

- Implantation losses (%):
% pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100
% post-implantation loss = [(Group number of implantation sites - number of offspring) / number of implantation sites] x 100
offspring) x 100

Live Birth and Viability Indices
- Live Birth Index (%) = (Number of offspring alive on Day 1/ number of offspring born) x 100
- Viability Index 1 (%) = (Number of offspring alive on Day 4/ number of offspring alive on Day 1) x 100
- Viability Index 2 (%) = (Number of offspring alive on Day 7/ number of offspring alive on Day 4) x 100
- Viability Index 3 (%) = (Number of offspring alive on Day 14/ number of offspring alive on Day 7) x 100
- Viability Index 4 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 14) x 100
- Viability Index 5 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 1) x 100

For further reproductive indices refert to 7.8.1 Toxicity to reproduction key, DuPont, 2011, rat, RL1.
Historical control data:
Historical control data for absolute and relative organ weight values, reproductive indices, fertility parameters and clinical observations were provided from Charles River (UK) Limited (2005-2008) and included in the study report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no treatment related deaths amongst maternal animals.

BODY WEIGHT AND FOOD CONSUMPTION
No adverse effects of treatment on mean bodyweight or bodyweight change was apparent in either generation for females. A significant increase in bodyweight gain during Days 1-21 in the low- and high-dose female P group was considered to be not treatment related.
No adverse effect of treatment on mean food consumption was apparent females. Food consumption of P females in the high-dose group was significantly lower during the last week of lactation. In mid-dose P females a higher food intake was observed during the last week of gestation and lactation.

ORGAN WEIGHTS
For P females of the high-dose group, absolute and bodyweight-relative thyroid weights were significantly higher. There were no significant differences in thyroid weights for F1 females or for males in either generation at this inclusion level. Given the earlier and potentially higher exposure of the F1 females to the test substance (due to low bodyweight at this stage) and the absence of any accompanying effect on thyroid weights for these animals, this finding was therefore considered to be incidental and of no toxicological significance. For P females receiving the mid and high-dose, absolute uterine weight was significantly lower; values when adjusted for the bodyweight were not significantly different from control and this finding was therefore considered to be coincidental and to reflect normal biological variation. In the low-dose P female group bodyweight-relative brain weight was significantly lower than control. In the absence of any dosage relationship, and any effect on absolute brain weight, this was considered to be incidental and unrelated to treatment.

GROSS PATHOLOGY
The majority of macroscopic necropsy findings of P females were limited to decedent animals; these deaths were considered to be unrelated to treatment.

HISTOPATHOLOGY
There were no histopathological findings observed for adults that were considered to indicate an adverse effect of the test substance at a dietary inclusion level of 25000 ppm.

POSITIVE CONTROL
Treatment at 10000 ppm DEPH was associated with clear signs of adult toxicity including notable effects on bodyweight and some organ weights. Reproduction of P animals was unaffected by treatment but shorter male ano-genital distance, increased male nipple counts and delays in attainment of sexual maturation suggested a possible endocrine disruption effect.

For further details refer to 7.8.1 Key, Danisco, 2009, rat, RL1

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 25 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 2 200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 25 000 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 342 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
>= 2 262 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
Survival of the offspring to weaning was unaffected at the dietary concentrations by either maternal treatment or by more direct treatment when the offspring consumed the diet as they approached weaning in both generations.

CLINICAL SIGNS (OFFSPRING)
No adverse effects were observed for the test substance treated animals.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on litter weights or bodyweight changes. For the F1 offspring at the high-dose litter weights were slightly lower between Days 14-21 (see Table 2 under “Any other information on results incl. tables”). For F2 offspring at the low and high-dose group bodyweight gain was slightly lower than control for both sexes between Day 7-14. These differences may reflect normal biological variation and were not considered to indicate an adverse effect of treatment.

SEXUAL MATURATION (OFFSPRING)
There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups. There was no adverse effect of treatment on mean ano-genital distance on Day 1 of age for either sex. For F1 males at the low-dose and F2 females at the mid-dose, mean ano-genital distance on Day 1 of age was slightly shorter (see Table 3 under “Any other information on results incl. tables”). These decreases were considered to reflect normal biological variation and were considered to be unrelated to treatment. There was no adverse effect of treatment on visible nipple counts on Days 11-15 of age for either sex in either generation. Only for F1 females at the high-dose visible nipple counts were marginally higher than control only at Day 11 of age, therefore this effect was considered to be incidental and unrelated to treatment (see Table 4 under "Any other information on results incl. tables").

GROSS PATHOLOGY (OFFSPRING)
The macroscopic abnormalities observed for both decedent and terminal kill offspring were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment with the test substance in either generation.

POSITIVE CONTROL (OFFSPRING)
Adverse effects on offspring survival and growth were apparent and offspring failed to thrive following separation from the parent female at weaning. Effects on brain, spleen, thymus and, for females, uterus weight were apparent for the F1 offspring. Necropsy revealed enlarged liver and kidneys, small testes, epididymides and seminal vesicles for male F1 animals.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1. Overall achieved intakes during each generation for test material treated groups.

Dietary Concentration

Males (mg/kg bw/day)
   F0   F1   

Females (mg/kg bw/day)
Maturation Gestation Lactation


 F0     F1     F0      F1    F0    F1

1500 ppm 82 109 106 135 101 108 231 238
6000 ppm 324 435 431 540 411 434 919 918
25000 ppm 1159 1342 1392 1493 1665 1697 3544 3596

Table 2. Table for reproductive performance.

Parameter

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Live Birth Index

F0-F1

F1-F2

 

99.3

99.6

 

98.2

98.3

 

98.4

99.5

 

98.4

99.5

Pre-Implantation Loss [%]

F0-F1

F1-F2

 

5.4 ± 4.6

5.5 ± 8.7

 

6.8 ± 7.4

4.9 ± 8.8

 

5.7 ± 4.0

5.4 ± 9.8

 

5.4 ± 6.1

1.7 ± 3.1

Post-Implantation Loss [%]

F0-F1

F1-F2

 

3.1 ± 6.5

7.0 ± 8.7

 

3.7 ± 5.5

4.2 ± 5.2

5.0 ± 7.4

4.9 ± 7.8

10.8 ± 16.2**

7.7 ± 13.8

Total Litter Weight (g)

F1 Day 1

F1 Day 14

F1 Day 21

F2 Day 1

F2 Day 14

F2 Day 21

 

95.1

389.5

600.0

87.3

363.2

564.2

 

89.6

362.6

567.4

101.0

384.1

599.3

 

92.4

368.8

584.8

95.6

368.9

570.4

 

88.9

343.7**

523.8**

98.0

360.0

555.2

Total number of Corpora Lutea

F0-F1

F1-F2

 

15.7 ± 1.6

15.3 ± 2.3

 

15.7 ± 2.7

17.2 ± 2.2

 

15.7 ± 1.8

16.5 ± 2.9

 

15.9 ± 2.0

16.3 ± 3.8

Total number of Implantation Sites

F0-F1

F1-F2

 

 

14.8 ± 1.7

14.4 ± 2.5

 

 

14.5 ± 2.7

16.3 ± 1.8

  

14.8 ± 2.0

15.4 ± 2.4

 

15.0 ± 1.7

16.0 ± 3.7

Total Number of Offspring Born

F0-F1

F1-F2

 

14.4 ± 2.1

13.5 ± 2.9

  

14.0 ± 2.6

15.5 ± 1.7

14.2 ± 2.4

14.6 ± 2.5

  

13.4 ± 1.7

14.8 ± 4.0

Sex ratio (% male at birth)

51.9

45.3

46.8

51.8

Table 3. Ano-Genital distance of offspring - group mean litter values.

Ano-genital distance (mm) on Day 1 post partum

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Males

F1

F2

3.12 ± 0.34

3.01 ± 0.33

2.95* ± 0.40

3.03 ± 0.33

3.12 ± 0.46

3.01 ± 0.33

3.13 ± 0.36

3.11 ± 0.31

Females

F1

F2

1.60 ± 0.34

1.47 ± 0.19

1.65 ± 0.44

1.51± 0.24

 

1.49 ± 0.31

1.33± 0.14**

 

1.67 ± 0.34

1.49± 0.22

p** ≤ 0.01

Table 4. Nipple counts for the F1 generation at Day 11 of age.

Group

Mean Nipple count at Day 11 of age

Male

Female

Control

0.0 ± 0.0

9.4 ± 1.8

Low dose group

0.1 ± 0.5

10.4 ± 1.5

Mid-dose group

0.0 ± 0.0

9.9 ± 1.5

High-dose group

0.1 ± 0.3

10.6 ± 1.4*

p* ≤ 0.05

Applicant's summary and conclusion

Conclusions:
The test substance had no effect on intrauterine development.