Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1996 to May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant and well documented study performed according to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
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): WITAMOL 110/LINPLAST 610 P
- Substance type: pure active substance
- Physical state: colourless liquid
- Lot/batch No.: 950608
- Storage condition of test material: in the dark at ambient temperature
- Other: Stability has been proven at the end of the study

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Area 57 of Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: (P) approx. 4 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 84-104 g; Females: 57-95 g
- Fasting period before study: no
- Housing: 2 animals per cage in polypropylene cages with stainless steel grid bottoms, mesh tops and food hoppers; mated females were housed individually in solid bottom cages (dams and their litters retained this type of cage until termination)
- Diet : Rat and Mouse Breeder Diet No. 3 SQC (Expanded) Fine Ground (ad libitum) by Special Diets Services Limited (SDS), Stepfield, Witham, Essex, UK
- Water: domestic mains water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 50 +/- 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: July 23, 1995 To: May 22, 1997

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly, at the end of the study at least every 14 days
- Mixing appropriate amounts with untreated diet
- Storage temperature of food: ambient temperature
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 7 nights
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility for max. 14 nights.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually. Sterilised white wood shavings were provided as bedding. White paper tissue was supplied to each mother for incorporation in the nest (this was replaced when it became soiled)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of triplicate samples for concentration and homogeneity on 5 occasions. An analytical method has been developed and validated. An internal standard was added and the samples were extracted with hexane. Aliquots of the extract were analysed by gas chromatography using flame ionisation detection.
Duration of treatment / exposure:
P animals were treated for 10 weeks prior to mating (commencing at approx. 6 weeks of age). Treatment continued throughout mating, gestation and lactation period until termination after weaning of these litters.
Selected F1 animals were treated for approx. 11 weeks after weaning, prior to mating. Treatment then continued for both sexes throughout the mating, gestation and lactation periods, until termination at the time of weaning of the F2 litters.
The selected F2 animals were treated until termination on the completion of the post-weaning assessments.
Frequency of treatment:
continuously with the diet
Details on study schedule:
- F1 parental animals not mated until 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 24 days of age.
- Age at mating of the mated animals in the study: approx. 14 weeks
- Selection of 1 male and 1 female per litter F2 pups on days 21-24 of lactation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
28 (P), 24 (F1)
Control animals:
yes, plain diet
Details on study design:
1000 ppm corresponds to 78 mg/kg bw/d male and 116 mg/kg bw/d female main dose
3000 ppm corresponds to 235 mg/kg bw/d male and 346 mg/kg bw/d female main dose
10000 ppm corresponds to 809 mg/kg bw/d and 1181 mg/kg bw/d female main dose

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 animals: one week prior to the first day of treatment, then weekly thereafter until the start of the mating period. Males continued weekly weighing until termination. Weights of females were also recorded on Days 0, 7, 14 and 20 of gestation, then on days 1, 7, 14 and 21 of lactation.
F1 parental animals: weekly from approx. 4 weeks of age, after the mating procedure same time schedule as F0 animals
Post-weaning F2: weekly until sacrifice

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, tubule stage and diameter (only control and high dose)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Body weight examination: Pre-weaning weighing en masse (sex separate) on days 1, 4, 7 and 14 of lactation. On day 21 of lactation the pups were weighed individually , and the total litter weight was also recorded.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
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 including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histological examination on testes and epididymides of control and high dose animals of P and F1 animals for qualitative and quantitative investigation of any effect on spematogenesis.
Weights of the following organs were recorded: testes, epididymides, seminal vesicles, coagulating gland, prostate gland, liver, kidneys
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and unselected F2 offspring were sacrificed at the time of weaning of F2 animals.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination)
GROSS NECROPSY (unselected F1 and F2 at weaning)
- 2 male and 2 female pups from each litter
- Gross necropsy consisted of external examinations including the cranial, thoracic, and abdominal cavities.
- Testes and body weight was recorded for 1 male in each litter
Statistics:
Where considered appropriate to assist with interpretation, statistical analysis was applied to determine the statistical significance of differences from Control. Organ weights were analysed by analysis of variance, and by analysis of covariance using the terminal body weight as the single covariate [Snedecor G. W. and w. G. Cochan (1980) Statistical Methods, 7th Edition. Iowa State University Press]. Pairwise comparisons between each treatment level and Control were performed using Fisher's F-protected Least Significant Differences.
Reproductive indices:
Fertility Index = (Number of pregnant females/siring males)/ Number Paired
Gestation Index = Number bearing live pups/Number Pregnant

Offspring viability indices:
Birth index = Total number of pups born (live and dead) / Number of implantation scars
Live Birth Index = Number of pups live on day 0 of lactation/ total number born (live and dead)
Viability Index = Number of pups live on day 4 of lactation / Number live on day 0
Lactation Index = Number of pups live on day 21 of lactation / Number of live on day 4
Overall Survival Index = Number of pups live on day 21 of lactation / Number of pups born (live or dead)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous 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)
There were no clinical findings observed which were considered to be related to treatment. One parental female of the high dose group was found dead in late lactation.

BODY WEIGHT (PARENTAL ANIMALS)
Weight gain of males of the high dose group in the P and F1 generations, and also in the retained F2 generation, was lower than control throughout the whole treatment period. Marginal differences of body weights of high dose females during lactation were considered too small to be attributable to treatment. There were no effects of treatment on weight gain at the lower dose levels. (table1)

FOOD CONSUMPTION
Food consumption of males of the high dose group in the P and F1 generations was slightly lower than that of control. A slight reduction in food consumption was observed in P females of the high dose group over day 7-21 of lactation and in F1 females throughout lactation. There was no effect on food consumption in females during the pre-mating period. Differences in the retained F2 generation were considered too small to be attributable to treatment. No effect on food consumption was observed in the low and mid dose groups. (table2)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
At all times the ratio between achieved dosages was essentially similar to the ratio of the dietary concentrations. As expected there was a steady decrease in achieved dosages with time over the treatment period for the males and during the pre-mating period for the females. During the gestation and lactation periods the values were generally increased and by the end of lactation values were up to 3 times higher than those during the pre-mating period.
For the periods where food consumption was measured, the mean achieved dosages were:
males: 78 (56-153), 235 (167-465) and 809 (591-1546) mg/kg bw/d
females: 116 (73-253), 346 (222-776) and 1181 (740-2666) mg/kg bw/d

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Values for tubular staging and diameter in the high dose group were similar to control values.
For testis weights and epididymides weights see ORGAN WEIGHTS.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no obvious effects of treatment on mating performance, fertility indices or the duration of gestation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In P, F1 and F2 adults, mean absolute testes weights were similar in all groups; following covariance analysis, the relative testes weights of F1 males in the high dose group was slightly greater than control with the difference attaining statistical significance.
Mean seminal vesicles weights were reduced in the high dose group, statistically significantly reduced for absolute weights in P, F1 and F2 adults and for relative weights in F1 and F2. In F2 adults absolute and relative seminal vesicles weights were also significantly reduced in the mid dose group.
Mean prostate weights were significantly lower in F1 males of the high dose group.
The mean absolute epididymides weights of high dose P adults was slightly but statistically significantly lower than control; however, this difference was no longer significant after adjustment for body weight. Due to the absence of similar findings in other generations the apparent difference was considered to reflect the lower body weights of these animals.
Mean absolute and relative liver weights were significantly increased in high dose females of all generations and in the mid dose group of P and F1 females. In the low dose group the absolute liver weights of F1 and F2 females were statistically significantly increased. After adjustment for body weight the statistical significance was only apparent for F1 females.
Among males, assessment effects on liver weights was complicated by body weight effects. After covariance adjustment, liver weights for P and F2 males of the mid and high dose group were statistically significantly increased, but weights for F1 males were similar to control. In all generations liver weights of males of the low dose group were similar to control.
Mean relative kidney weights were significantly greater than control in P and F1 females of the mid and high dose group. In F1 females there was also a slight statistically significantly increase in the low dose group. In males relative kidney weights in mid and high dose group of the F1 generation were increased, but similar increases were not seen in the other generations. Apparent reductions in absolute kidney weights, which were no longer present after adjustment for body weight, were considered to reflect the lower body weights. (table 3)

GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy findings in the majority of P, F1 and F2 males of the high dose group included discoloured, enlarged and pale liver with prominent lobulation and/or pale/dark focus. Pale liver foci were also observed for occasional males in the mid dose group and for occasional females in the mid and high dose group. The other necropsy findings were considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histology in the high dose group revealed numerous findings affecting the liver in the majority P and F1 males; these comprised of generalised cellular change, basophilic foci(us), vacuolated focus, eosinophilic cell focus, clear cell focus, bile duct hyperplasia and Kupffer-cell pigmentation. The remaining findings were considered not to be related to treatment.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive effects/fertility
Effect level:
167 - 776 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: reduced pup weights, marginal delay in sexual maturity (3000 ppm)
Remarks on result:
other: Generation: F1, F2 (migrated information)
Dose descriptor:
LOAEL
Remarks:
reproductive effects/fertility
Effect level:
591 - 2 666 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: reduced pup survival and weights, marginal delay in sexual maturiry (10000 ppm)
Remarks on result:
other: Generation: F1, F2 (migrated information)

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:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
effects observed, treatment-related

Details on results (F1)

LITTER SIZE AND VIABILITY (OFFSPRING)
The mean number of implant sites were similar in all groups.
In the high dose there was a slight increase in the number of P and F1 animals losing more than 1 pup during Days 4 and 21 of lactation, with a corresponding slight reduction in the litter size over that period. In the mid and low dose groups litter size and survival were similar to control. (tables 4 and 5)

CLINICAL SIGNS (OFFSPRING)
There were no clinical findings observed which were considered to be related to treatment. The findings in F2 animals comprised of one incidence of scabbing and one incidence of swollen tail.

BODY WEIGHT (OFFSPRING)
In the high dose group litter and pup weight were lower than control in both generations. Pup weights in mid dose group were similar to control, but litter weights were slightly lower, reflecting an incidentally lower litter size. In low dose group litter weight and pup weight were essentially similar to control. (tables 6 and 7)

SEXUAL MATURATION (OFFSPRING)
In the high dose group the mean age of preputial separation was marginally greater than control, although body weights at the time of separation were similar. Considering the 2 generations together, the mean age at separation at the mid and low dose levels was similar to control.
In F1 animals of mid and high dose group vaginal opening occurred slightly later than that in controls; body weights at vaginal opening in these groups were marginally higher than that of controls. In the F2 animals there were no differences in the age of vaginal opening. (table 8)

ORGAN WEIGHTS (OFFSPRING)
Testes weights of weanlings were slightly lower than control in the high dose group, with the values for F1 weanlings attaining statistical significance (table 9).

GROSS PATHOLOGY (OFFSPRING)
Necropsy findings in the majority of F1 and F2 males of the high dose group included discoloured, enlarged and pale liver with prominent lobulation and/or pale/dark focus. Pale liver foci were also observed for occasional males in the mid dose group and for occasional females in the mid and high dose group. The other necropsy findings were considered to be incidental.

Effect levels (F1)

Dose descriptor:
LOAEL
Generation:
F1
Effect level:
56 - 253 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: increased liver and kidney weights (1000 ppm)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Group mean body weights (g) of males at selected time points (mean values and standard deviations)
Group  Control 1000 ppm 3000 ppm 10000 ppm
P generation week of treatment
0 222 ± 19 218 ± 19 214 ± 15 213 ± 19
4 408 ± 31 403 ± 33 392 ± 27 359 ± 39
8 495 ± 42 490 ± 51 477 ± 37 421 ± 54
12 539 ± 46 541 ± 60 522 ± 36 456 ± 53
16 588 ± 47 589 ± 68 565 ± 39 497 ± 58
F1 generation week of age

4

106 ± 13

107 ± 18

107 ± 17 

106 ± 16

8 332 ± 24 334 ± 34 334 ± 28 316 ± 28

12

436 ± 39

436 ± 41

432 ± 42 

395 ± 40

18 523 ± 53 520 ± 45 527 ± 48 460 ± 46
22 570 ± 54 559 ± 50 569 55 492 ± 48
F2 generation week of age
4 114 ± 14 112 ± 13 112 ± 18 106 ± 15
5 171 ± 19 168 ± 15 168 ± 24 161 ± 20
6 233 ± 23 225 ± 18 230 ± 17 219 ± 23
Table 2: Group mean food consumption (g/rat/d) at selected time points
Group  Control 1000 ppm 3000 ppm 10000 ppm
P males week of treatment
0 25.3 24.9 24.7 24.4
4 32.2 31.9 30.9 28.7
8 31.5 32.1 30.9 28.9
12 29.8 30.1 29.6 27.5
16 32.6 32.6 31.6 29.8
F1 males week of age
5 20.8 20.8 21.2 21.1
8 32.3 32.6 32.3 31.5
12 31.5 30.6 30.5 28.7
18 30.3 29.5 30.3 28.0
22 31.1 31.3 31.4 28.8
P females Day of lactation
0 - 7 44.5 47.1 46.4 48.6
7 - 14 76.4 75.3 76.1 69.7
14 - 21 87.7 87.6 85.4 76.6
F1 females Day of lactation
0 - 7 45.7 48.1 46.7 36.1
7 - 14 75.4 77.6 75.4 55.3
14 - 21 87.9 90.0 91.8 62.6
Table 3: Selected relative organs weights (g) (mean values and standard deviations)
Group Control 1000 ppm 3000 ppm 10000 ppm
P males
Liver 19.85 ± 0.43 20.46 ± 0.43 21.69 ± 0.42** 22.80 ± 0.48***
P females
Liver 16.67 ± 0.37 16.95 ± 0.39 18.44 ± 0.38** 22.64 ± 0.37***
Kidney 2.65 ± 0.04 2.70 ± 0.05 2.84 ± 0.04** 2.92 ± 0.04***
F1 males
Testis 3.61 ± 0.06 3.66 ± 0.06 3.68 ± 0.06 3.91 ± 0.07**
Seminal vesicles 2.583 ± 0.112 2.673 ± 0.110 2.442 ± 0.111 2.151 ± 0.123*
Prostate gland 0.88 ± 0.04 0.89 ± 0.04 0.83 ± 0.04 0.69 ± 0.05**
Kidneys 3.82 ± 0.08 3.93 ± 0.08 4.06 ± 0.08 4.30 ± 0.09***
F1 females
Liver 15.00 ± 0.48 16.49 ± 0.49* 17.07 ± 0.48** 20.52 ± 0.48***
Kidney 2.44 ± 0.04 2.57 ± 0.04* 2.59 ± 0.04** 2.62 ± 0.04**
F2 males
Seminal vesicles 0.551 ± 0.024 0.503 ± 0.024 0.462 ± 0.024** 0.472 ± 0.023*
Liver 14.44 ± 0.34 14.25 ± 0.34 15.41 ± 0.34* 16.02 ± 0.33**
F2 females
Liver 9.77 ± 0.18 9.77 ± 0.18 10.18 ± 0.19 10.80 ± 0.18***
* p < 0.05; ** p < 0.01; *** p < 0.001
Table 4: Pup survival during lactation days 4 -21 (No. of dams losing > 1 pup)
Group Control 1000 ppm 3000 ppm 10000 ppm
P dams  2 0 2 5
F1 dams 1 1 1 6
Table 5: Litter size during lactation (mean No. of live pups and standard deviations)
Group Control 1000 ppm 3000 ppm 10000 ppm
F1 litter Day 0 of lactation 14.6 ± 2.1 13.0 ± 2.3 13.7 ± 3.3 13.5 ± 2.3
Day 4 of lactation 13.6 ± 1.8 12.3 ± 2.2 12.7 ± 3.4 12.7 ± 2.1
Day 7 of lactation 13.3 ± 1.9 12.2 ± 2.2 12.5 ± 3.6 12.5 ± 2.0
Day 14 of lactation 13.1 ± 1.9 12.0 ± 2.2 12.3 ± 3.6 12.1 ± 2.0
Day 21 of lactation 13.1 ± 1.9 12.0 ± 2.3 12.3 ± 3.7 12.0 ± 2.0
F1 litter Day 0 of lactation 14.3 ± 2.4 14.3 ± 1.9 13.5 ± 3.2 13.1 ± 3.8
Day 4 of lactation 13.2 ± 2.9 13.3 ± 2.5 12.7 ± 3.2 12.2 ± 4.0
Day 7 of lactation 13.1 ± 2.9 13.3 ± 2.5 12.5 ± 3.1 12.0 ± 4.1
Day 14 of lactation 13.4 ± 2.9 13.2 ± 2.5 12.3 ± 3.1 11.4 ± 3.9
Day 21 of lactation 13.0 ± 2.9 13.1 ± 2.6 12.3 ± 3.1 11.4 ± 3.9
Table 6: Litter weights (group mean values and standard deviations)
Group Control 1000 ppm 3000 ppm 10000 ppm
F1 litter Day 1 of lactation 89 ± 12 83 ± 13 82 ± 19 86 ± 14
Day 4 of lactation 124 ± 20 119 ± 26 114 ± 30 119 ± 16
Day 7 of lactation 178 ± 30 173 ± 36 163 ± 44 167 ± 20
Day 14 of lactation 345 ± 44 335 ± 56 320 ± 76 295 ± 32
Day 21 of lactation 548 ± 76 543 ± 93 498 ± 121 451 ± 56
F2 litter Day 1 of lactation 73 ± 37 90 ± 13 82 ± 15 78 ± 21
Day 4 of lactation 102 56 127 ± 24 120 ± 23 108 ± 34
Day 7 of lactation 152 ± 82 189 ± 30 179 ± 33 156 ± 50
Day 14 of lactation 289 ± 154 358 ± 44 336 ± 51 274 ± 79
Day 21 of lactation 456 ± 243 568 ± 63 353 ± 76 421 ± 120
Table 7: Pup weights (g) (mean of litter mean values and standard deviations)
Group Control 1000 ppm 3000 ppm 10000 ppm
F1 generation
males Day 1 of lactation 6.5 ± 0.6 6.7 ± 0.7 6.6 ± 0.8 6.8 ± 0.7
Day 21 of lactation 43.1 ± 6.1 46.9 ± 5.8 42.6 ± 7.2 39.3 ± 6.6
females Day 1 of lactation 6.1 ± 0.6 6.3 ± 0.6 6.1 ± 0.6 6.3 ± 0.5
Day 21 of lactation 41.6 ± 6.1 44.7 ± 5.8 41.0 ± 7.2 37.4 ± 6.2
F2 generation
males Day 1 of lactation 6.6 ± 0.8 6.6 ± 0.6 6.6 ± 1.0 6.5 ± 1.1
Day 21 of lactation 45.0 ± 6.3 45.2 ± 6.5 46.1 ± 9.0 40.0 ± 9.2
females Day 1 of lactation 6.3 ± 0.8 6.3 ± 0.7 6.3 ± 1.1 6.0 ± 0.8
Day 21 of lactation 42.9 ± 6.3 43.5 ± 6.2 44.3 ± 9.1 37.2 ± 7.8
Table 8: Sexual maturation (mean values and standard deviations)
Group Control 1000 ppm 3000 ppm 10000 ppm
Age at preputial separation (days)
F1 males 44.9 ± 1.7 45.0 ± 2.3 45.7 ± 1.9 46.3 ± 2.2
F2 males 45.0 ± 2.6 45.5 ± 2.4 44.8 ± 2.1 46.0 ± 2.0
Weight at preputial separation (g)
F1 males 220 ± 17 221 ± 18 223 ± 15 221 ± 18
F2 males 222 ± 19 218 ± 19 222 ± 25 214 ± 22
Age at vaginal opening (days)
F1 females 35.8 ± 3.2 35.1 ± 4.2 374 ± 3.2 37.8 ± 4.2
F2 females 35.6 ± 2.8 35.6 ± 2.7 35.0 ± 2.4 36.2 ± 2.8
Weight at vaginal opening (g)
F1 females 122 ± 19 117 ± 23 127 ± 15 128 ± 26
F2 females 122 ± 16 119 ± 15 121 ± 17 120 ± 15
Table 9: Relative testis weights (g) of unselected weanlings (mean values and standard deviations)
Group Control 1000 ppm 3000 ppm 10000 ppm
F1 generation 0.42 ± 0.01 0.40 ± 0.01 0.41 ± 0.01 0.38 ± 0.01**
F2 generation 0.47 ± 0.01 0.45 ± 0.01 0.45 ± 0.01 0.43 ± 0.01
** p < 0.01

Applicant's summary and conclusion

Conclusions:
1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters caused reproductive effects as reduced pup survival and weights, and marginal delay in sexual maturity at the 10000 ppm. Effects on reproductive organs, liver and kidney were essentially similar in all generations and were therefore not classed as specific reproductive effects. The NOAEL for reproductive toxicity is therefore considered to be 3000 ppm.
Executive summary:

In a two-generation fertility study with 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters reproductive effects occurred, seen as reduced pup survival and weights, and a marginal delay in sexual maturity at a treatment level of 10000 ppm (corresponding to an intake in the range of 591 - 2666 mg/kg bw/d, mean concentration in females 1181 mg/kg bw/d, mean concentration in males 809 mg/kg bw/d ). Effects on reproductive organs, liver and kidney were essentially similar in all generations and were therefore not classed as specific reproductive effects. The NOAEL for reproductive toxicity was therefore considered to be 3000 ppm (corresponding to a mean daily intake of 167 -776 mg/kg bw/d, mean concentration in males 235 mg/kg bw/d and mean concentration in females 346 mg/kg bw/d. It was not possible to identify a no effect level in this study, although at 1000 ppm (corresponding to an intake in the range of 56 -253 mg/kg bw/d) were confined to increased liver and kidney weights among F1 females (LOAEL 1000 ppm).