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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to an equivalent of OECD 415 and under GLP conditions. However, relevant appendices in the report describing the macroscopy of P and F1 and a table describing the histopathology of F1 are missing, as well as a table with clinical observations. Additionally, the certificate of analysis for the diet and all individual data are missing.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
no histopathology of the F1 was performed
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-ethylhexyl diphenyl phosphate (EHDP)
- Physical state: Liquid
- Storage condition of test material: Glass container at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Olac Ltd, bicester, Oxon, UK
- Age at study initiation: (M) 14-15 weeks, (F) 11 weeks
- Weight at study initiation: (M) 280-282 g, (F) 208-212 g
- Housing: (M) groups of 2 according to sex and treatment, (F) groups of 2 according to sex and treatment and individually after mating
- Diet (e.g. ad libitum): Nutritionally adequate diet
- Water (e.g. ad libitum): Domestic mains tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 45-70
- Air changes (per hr): Min. of 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet
- Storage temperature of food: 2-6 °C
Details on mating procedure:
- M/F ratio per cage: 1:2
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually

After unsuccesful mating (female not in oestrous) the female was left caged with the male until signs of succesful mating were observed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diets were analysed in duplicate for content of test article and were also tested for stability. Efficiency of mixing test article with rodent diet was proven in a previous study.
Duration of treatment / exposure:
Males: 70 days prior to mating, during mating and up to the end of the study
Females: 21 days prior to mating, during mating, during pregnancy, rearing and up to the end of the study
Frequency of treatment:
Continuously (diet)
Details on study schedule:
Not relevant
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.2, 0.4 and 0.8% 2-EHDP
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
M: 132, 259 and 490 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
F: 144, 293 and 536 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
16 males and 32 females per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on results from previous repeated dose toxicity studies
- Rationale for animal assignment: By use of random number tables
Positive control:
Not relevant

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: Daily
- Cage side observations: Appearance of animal and excreta, behaviour

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Weekly

BODY WEIGHT
- Time schedule for examinations: (M) Weekly, except around day 1 of treatment (daily), (F) Daily around day 1 of treatment, weekly prior to and daily during pregnancy, on day 1, 4, 7 and 14 after parturition

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption: Yes, measured between intervals of weighing (except during mating)
- Compound intake: Yes, calculated from food consumption

WATER CONSUMPTION
- Time schedule: Measured between the intervals of weighing, except during mating
Oestrous cyclicity (parental animals):
Not performed
Sperm parameters (parental animals):
Parameters examined in parental males of the high dose and control group: testis weight, epididymis weight (as part of organ weight analysis)
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight (gain), abnormalities

GROSS EXAMINATION OF DEAD PUPS: Performed, but no data available
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations in all animals
- The following tissues were retained:
adipose tissue, adrenal grlands, artery (aorta), bladder (urinary), brain, caecum, colon, cervix uteri, diaphragm, epididymis, eye, femur, harderian gland, heart, kidneys, liver, lungs, lymph nodes, mammary gland, nasal bones, sciatic nerve, oesophagus, ovaries, pancreas, pinnae, pituitary, prostate, rectum, salivary gland, seminal vesicles, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, uterine horns, vagina, vein, any other abnormal tissue.

ORGAN WEIGHTS
The organs indicated below were weighed from all animals:
Adrenal glands, brain, caecum, gonads, heart, liver, kidney, spleen.

HISTOPATHOLOGY
The tissues indicated below and tissues showing macroscopic abnormalities were prepared for microscopic examination (only from all high dose and control animals):
Cervix uteri, epididymes, liver, ovaries, pituitary, prostate, seminal vesicles, testes, uterine horns, vagina.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 21 days of age.
- All animals were subjected to postmortem macroscopic examinations, while tissues were retained, organ weight were measured and histopathology was performed only for randomly chosen male and female animals from each litter.

GROSS NECROPSY
- Gross necropsy consisted of external examinations in all pups
- The following tissues were retained from randomly chosen male and female animals from each litter:
adipose tissue, adrenal grlands, artery (aorta), bladder (urinary), brain, caecum, colon, cervix uteri, diaphragm, epididymis, eye, femur, harderian gland, heart, kidneys, liver, lungs, lymph nodes, mammary gland, nasal bones, nerve, oesophagus, ovaries, pancreas, pinnae, pituitary, prostate, rectum, salivary gland, seminal vesicles, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, uterine horns, vagina, vein, any other abnormal tissue.

ORGAN WEIGHTS
The organs indicated below were weighed from the randomly chosen male and female animals from each litter:
Adrenal glands, brain, caecum, gonads, heart, liver, kidney, spleen.

HISTOPATHOLOGY
Not performed
Statistics:
- Kolmogorov-Smirnov test
- Bartlett's test
- Analysis of variance
- Least significant difference test
- Wilcoxon Mann-Whitney test
- T-test
- Fisher's exact test
Reproductive indices:
- Mating rate
- Fertility rate (gestation index)
Offspring viability indices:
No data

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
1 male (no. 33) and 1 female (no. 181) died during the study. Unknown from which group: Relevant appendix of report missing!

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
Significant effects on body weight and food consumption were observed at several timepoints throughout the study (see table in "Any other information on results incl. tables"). Only for the effect on body weight a dose-related response was observed.

Females:
Significant effects on body weight and food consumption were observed at several timepoints throughout the study (see table in "Any other information on results incl. tables"). Both effects showed a dose-related response.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Relative testes weight was significantly increased for the mid and high dose group males. No effect on the epididymes weight was observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant difference between the test group and the control group was observed in precoital interval, mating index, duration of gestation and gestation index.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In all test groups and for both sexes significant increases of absolute and relative organ weight were observed (see table in "Any other information on results incl. tables").

GROSS PATHOLOGY (PARENTAL ANIMALS)
No data: Relevant appendix of report missing!

HISTOPATHOLOGY (PARENTAL ANIMALS)
Significant effects were observed in the livers of males of the highest dose group. In females, significant effects were observed in liver, ovaries and uterus.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
132 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Significant effects on relative organ weights in the lowest dose group
Dose descriptor:
LOAEL
Effect level:
144 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
during pre-mating and pregnancy
Sex:
female
Basis for effect level:
other: Significant effects on relative organ weights in the lowest dose group

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No data on number of live births: Relevant appendix of report missing!
Mean number of pups per litter was significantly lower for the mid and high dose groups from PD1 up to PD21 (see table in "Any other information on results incl. tables").

CLINICAL SIGNS (OFFSPRING)
No data on clinical signs: Relevant appendix of report missing!

BODY WEIGHT (OFFSPRING)
Significantly reduced for the mid and high dose group pups (see table in "Any other information on results incl. tables").

ORGAN WEIGHTS (OFFSPRING)
In all test groups significant increases of absolute and relative organ weight were observed (see table in "Any other information on results incl. tables"). The decreases in the absolute organ weight can be considered related to the decrease in body weight in the mid and high dose group, however, this does not explain the effect on relative organ weights. The organ weight effects may have been caused by direct developmental toxicity or by the poor general condition (body and organ weight effects) of the dams.

GROSS PATHOLOGY (OFFSPRING)
No data: Relevant appendix of report missing!

Effect levels (F1)

Dose descriptor:
LOAEL
Remarks:
developmental
Generation:
F1
Effect level:
144 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Significant effects on relative organ weights (liver) in the lowest dose group

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Test article:diet mixtures were found to be stable in closed containers for a minimum of 21 days at room temperature and a minimum of 28 days at 4 °C. Diets stored in open containers under animals room conditions were stable for a minimum of 7 days. Appendix including this information is missing in the report.

RESULTS OF TEST DOSING GROUPS
Control Low (0.2%) Medium (0.4%) High (0.8%)
ANALYSES
Actual concentration 0 mg/kg bw/day M: 132 mg/kg bw/day M: 259 mg/kg bw/day M: 490 mg/kg bw/day
F: 144 mg/kg bw/day (during pre-mating and pregnancy) F: 293 mg/kg bw/day (during pre-mating and pregnancy) F: 536 mg/kg bw/day (during pre-mating and pregnancy)
Stability Relevant appendix of report missing
Homogeneity Relevant appendix of report missing
Test substance intake Relevant appendix of report missing
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL - PARENTAL DATA (P - MALE)
Number of animals 16 16 16 16
Mortality x x x x
Body weight x x *Decreased from day 0-119 *Decreased from day 0-119
Food consumption x x x x
Water consumption x x x x
Clinical signs Relevant appendix of report missing
Relative organ weights x *Decrease in spleen weight and increase in liver, kidney and empty caecum weight *Increase in liver and testes weight *Increase in adrenal, brain, kidney, liver, testes and empty caecum weight
Macroscopy Relevant appendix of report missing
Microscopy x Not examined Not examined Liver: *Increase in cytoplasmic vacuolisation (n=16), hypertrophy (n=16) and lipofuscin (n=3)
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL - PARENTAL DATA (P - FEMALE)
Number of animals 32 32 32 32
Mortality x x x x
Body weight x x *Decreased from day 18-21 of pregnancy and day 7-21 post-partum *Decreased during pre-mating, pregnancy (except on day 1) and post-partum
Food consumption x x *Decreased on day 1-21 postpartum *Decreased from day 0-21 during pre-mating, day 0-20 during pregrancy and day 1-28 post-partum
Water consumption x *Decreased from day 14-21 post-partum *Decreased on day 7-21 post-partum *Decreased on day 1-28 post-partum
Clinical signs Relevant appendix of report missing
Relative organ weights x *Increase in adrenal and liver weight *Increase in adrenal, brain, kidney and liver weight, decrease in spleen weight *Increase in adrenal, brain, kidney, liver, ovary and empty caecum weight, decrease in spleen weight
Macroscopy Relevant appendix of report missing
Microscopy Uterus: haemosiderin uterine wall (n=14) Not examined Not examined Liver: *Increase in cytoplasmic vacuolisation (n=26), hypertrophy (n=19), bile duct hyperplasia (n=31). Ovaries: *Increase in hypertrophy/luteinisation stroma (n=32). Uterus: *Increase in haemosiderin uterine wall (n=22)
Precoital interval (duration) 3.8 3.6 3.3 4.5
Mating index (%) 100 100 100 100
Duration of gestation 21.9 21.8 21.9 21.9
Gestation index (%) 72 88 81 88
Number of litters 23 28 26 28
 
OFFSPRING TOXICITY (F1)
Litter size (numbers) 270 306 255 271
Mean litter size (day 1) 11.7 10.9 9.8 9.7
Mean litter weight 76.2 72.2 62.8* 57.2*
Sex ratio (M/F) 0.45 0.51 0.52 0.48
Mean litter size (day 21) 10.9 10.5 9.5 7.4
Post natal survival until day 21 (%) 93 95.8 96.9 68.3
Postnatal growth (BW gain) x x *Decreased from day 1-21 *Decreased from day 1-21
Relative organ weights (male) x *Increase in liver weight *Increase in adrenal, brain and liver weight, decrease in heart and spleen weight *Increase in adrenal, brain, heart and liver weight, decrease in spleen weight
Relative organ weights (female) x *Increase in liver weight, decrease in heart and spleen weight *Increase in adrenal, brain and liver weight, decrease in heart and spleen weight *Increase in adrenal, brain and liver weight, decrease in ovary and spleen weight
Macroscopic observations Relevant appendix of report missing
Microscopic observations No data (table missing in report)
x = no effects (as compared to control group)
* = significant effect (as compared to control group)

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, a developmental LOAEL of 144 mg/kg bw/day was established based on significant effects on the organ weights of F1 pups of the low dose group. The parental LOAEL was established to be 132 and 144 mg/kg bw/day for males and females, respectively, based on significant effects on relative organ weights in the low dose group.
Executive summary:

The study was conducted according to an equivalent of OECD 415 and used to determine effects of Santicizer 141 on the fertility and reproduction of parents and development of offspring. Male and female rats were treated with the test article prior to mating (M: 70 days, F: 21 days), during mating, during pregnancy and up to lactation (females only) with 0, 0.2, 0.4 or 0.8% Santicizer 141 in food (corresponding to 132, 259 and 490 mg/kg bw/day for males and 144, 293 and 536 mg/kg bw/day for females).

No mortality was observed for the parental treatment groups, but significant effects on primarily food consumption and relative organ weights were noted for the low dose group. Additional significant effects observed in the high dose groups were related to water consumption, body weight and histopathology (only high dose group examined). For the low dose group pups, only a statistical significant effect on relative organ weights was observed. Additional significant effects on postnatal weight gain and post natal survival were noted for the mid and high dose group.

Under the conditions of this study, a developmental LOAEL of 144 mg/kg bw/day was established based on a decrease in postnatal survival and litter size. The parental LOAEL was established to be 132 and 144 mg/kg bw/day for males and females, respectively.

The effects in P and F1 at the lowest dose level were considered minimal for this study, but when considering the (comparable) effects observed in the available 90-day repeated dose toxicity study (in which lower dose levels are tested), it is indicated that these observed effects are of toxicological relevance. The NOAEL is expected to be lower than the doses tested in this study and therefore the LOAEL was set at the lowest dose level.