Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The source substance 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 (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 is 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).

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
August 1996 to May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to the structural similarities and consistent trend in toxicokinetic and (eco)toxicological behaviour, the selected source substances are considered suitable and systemic human health effects and ecotoxicological effects can be directly read-across in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no
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
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
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
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)
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed
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.
Key result
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)
Key result
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)
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
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.

Key result
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)
Key result
Reproductive effects observed:
not specified
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
Conclusions:
The source substance 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 the source substance 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).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
235 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A two-generation study has been undertaken on a structurally related substance, phthalic acid di-n-hexyl, di-n-octyl, n-decyl ester which has C-chain lengths of C6, C10 compared to 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters C8 and C10. In this study reproductive effects occurred, seen as reduced pup survival and weights, and a marginal delay in sexual maturity at a treatment level of 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 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).

On the basis of all evaluated data, the similarity of the source substance is justified on basis of similar composition, structural similarity, the physico-chemical properties, toxicokinetics and toxicological profiles and supported by various QSAR methods. Therefore the read-across approach is used to fill data gaps. There is convincing evidence that the substances have an overall common category profile in regard to this endpoints. The substances have the same basic structure (diester of 1,2-Benzenedicarboxylic acid), with almost identical and overlapping alkyl chains. All of the substances (the target substance 1,2-Benzenedicarboxylic acid, C8 -10 -alkyl esters as well as the read-across substance) are esters. The target substance is a mixture of different isomers, with ca. 21 % dioctyl phthalate and ca. 28 % didecyl phthalate and ca. 48 % decyl octyl phthalate. Source substance 1,2-Benzenedicarboxylic acid, C6-10-alkyl esters (CAS-number 68515-51-5), is also a mixture of different isomers, with ca. 33 % dioctyl phthalate and ca. 18 % hexyl octyl phthalate and ca. 30 % decyl octyl phthalate and ca. 7.5 % didecyl phthalate. Therefore, the isomers of the target substance are also constituents of the read-across substance 1. Therefore it is likely that the same is valid for the test substance.

A similar study has been undertaken on a structurally related substance,1,2 benzene dicarboxylic acid , di C9-C11 alkyl esters which has C-chain lengths of C9, C11 compared to 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters C8 and C10. In this study no adverse effects upon fertility or reproductive performance when rats were exposed to diets containing up to 10000 ppm, corresponding to a mean daily intake of at least 650 mg/kg/day, through two successive generations. D911P fed through two generations had no adverse effect on the seminology parameters investigated and no histopathological effects on the reproductive organs.


Short description of key information:
Fertility: NOAEL - 235 mg/kg/day (similar substance)

Effects on developmental toxicity

Description of key information
Developmental toxicity in rats (Analogue approach) and rabbits: NOAEL - 1000 mg/kg/day 
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
August to November 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
common functional group(s)
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted May 12, 1981
Deviations:
yes
Remarks:
room temperature of 20 +/- 2 °C instead of 22 +/- 2 °C; acclimation period of 3-5 days instead of at least 5 days; acclimation period after mating
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: approx. 210 g
- Fasting period before study: no
- Housing: single in polypropylene cages with stainless steel grid bottoms and mesh tops
- Diet: Rat and Mouse Breeder Diet No. 3 (Expanded) SQC (by Special Diets Services (SDS) Limited, Stepfield, Witham, Essex, UK) ad libitum
- Water (e.g. ad libitum): domestics mains water adlibitum
- Acclimation period: 3-5 days prior dosing

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: October 20, 1995 To: November 6-8, 1995
Route of administration:
oral: gavage
Vehicle:
maize oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Mixing of requisite test material with correct quantity of vehicle in glass container by gentle manual inversion. The dose solutions were prepared freshly for the first 5 days of the dosing period. For the remaining 8 days of dosing, a single batch was prepared for each dose level, and a suitable aliquot from each batch was dispensed daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): total volume applied: 5 ml/kg
- Lot/batch no. (if required): no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Triplicate samples were taken from all dosing solutions on 2 occasions, the first and fifth day of dosing period. These samples were analysed for concentration and homogeneity.
Details on mating procedure:
The female rats were delivered time-mated by the supplier. The delivery consisted of 3 subbatches mated over 3 successive days. On delivery, one batch was on day 1 of gestation, a second batch on day 2 and a third on day 3 of gestation (Day of mating = day 0 of gestation).
No more than 2 study females were inseminated by the same male.
Duration of treatment / exposure:
days 6 to 16 of gestation
Frequency of treatment:
daily
Duration of test:
until day 20 of gestation
Remarks:
Doses / Concentrations:
100 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, with particular attention being paid to the period 1-2h after dosing


BODY WEIGHT: Yes
- Time schedule for examinations: days 4, 6-17 and 20 of gestation


FOOD CONSUMPTION : Yes, from day 3 of gestation
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: macroscopic examination of thoracic and abdominal contents

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: Yes
- Number of late resorptions: Yes
- Other: position of implantation sites

Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
-Other: sex of foetuses, weight of live foetuses

Statistics:
Body weight gain:
Parametric analysis of variance [Snedecor, GW and Cochran, WG (1980) Statistical Methods, 7th Edition, 213-254 and 365-392, Iowa State University Press.] Pairwise comparisons between each treatment level and control using Dunnett's test [Dunnet, CW (1964) Biometrics, 20, 482-491.] Where there was significant heterogeneity of variance, a log or square root transformation was used in an attempt to stabilise the variances. Where transformation failed to stabilise the variances, the Kruskal-Wallis test was used instead [Hollander, M and Wolfe, DA (1973) Non-parametric Statistical Methods, John Wiley and Sons, 114-137.]

Other parameters:
interpretation based on inspection of the individual and group values (formal statistical analysis not considered useful to conduct).

Historical control data:
no data
Clinical signs:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical observations:
Slight increase in the incidence of piloerection at 1000 mg/kg bw/d (see table 1)

Body weight performance:
At 1000 mg/kg bw/d slight increase in weight gain over days 6-17 of gestation, principally between days 13-17, with the increase over days 6-17 just failing to achieve statistical significance (P> 0.05). At 100 and 500 mg/kg/d, mean weight gains were marginally greater than control, but the differences were considered too small to be attributed to treatment. (See table 2)

Food consumption:
Slight increase in food consumption from day 10 of gestation in high dose group. In low and mid dose group food consumption was essentially similar to that of the controls. (See table 3)

Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Foetal abnormalities and variants:
Increased incidences of foetuses with 14th ribs in mid and high dose groups; the incidence in low dose group was similar to control (see table 4).

Increased incidence of foetuses with retarded sternebrae in the high dose group compared with control. In the low and mid dose groups the incidences of retarded sternebrae were not noticeable different from Control. (See table 5)



Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: supernumerary rib
Key result
Developmental effects observed:
not specified

Clinical Observations:

Table1:

Observation/finding

Dose level [mg/kg bw/d]

0

100

500

1000

Brown staining nasal region

1

1

0

2

Lower teeth cut; upper teeth broken

0

1

0

0

Wet/stained vagina

0

0

1

1

Soft faeces

0

0

0

1

Lump upper caudal region

1

0

0

0

Scabbing

1

1

0

0

Unkempt coat

0

0

1

0

Hairloss

0

0

0

1

Piloerection

0

1

2

5

Aggressive/agitated behaviour

0

0

1

1

Irregular respiration

0

0

0

1

Thin

0

1

0

0

Connected spleen and left kidney

1

0

0

0

Distended uterine horn

0

0

1

0

Body weight performance:

Table 2: Group mean body weight (g) ± Standard Deviation (pregnant animals only)

Day of Gestation

Dose level [mg/kg bw/d]

0

100

500

1000

4

225 +/- 10

227 +/- 11

224 +/-11

230 +/- 8

6

242 +/- 9

243 +/- 9

241 +/- 13

247 +/-9

9

259 +/- 11

262 +/- 10

260 +/- 15

263 +/- 9

13

294 +/- 13

297 +/- 12

295 +/- 18

299 +/-11

17

335 +/- 16

342 +/- 13

341 +/- 28

348 +/- 14

20

386 +/- 24

394 +/- 19

389 +/- 36

397 +/-18

Gain Days 6-17

93 +/- 11

99 +/- 10

99 +/- 19

102 +/-8

% of control

-

106

106

110

            Means are based on 17-25 animals

Food consumption:

Table 3: Group mean food consumption (g) ± Standard Deviation (pregnant animals only)

Day of Gestation

Dose level [mg/kg bw/d]

0

100

500

1000

3

28

28

28

28

4

26

27

25

27

5

28

28

28

29

6

25

24

25

26

7

28

28

29

29

8

28

28

29

29

9

30

29

30

31

10

30

32

32

33

11

32

32

32

35

12

31

32

32

34

13

31

31

31

34

14

33

33

34

36

15

35

35

34

36

16

34

34

35

36

17

38

37

36

39

18

34

36

35

37

19

28

29

29

31

Total Mean Consumed Days 6-16

337

338

343

359

% of Control

-

100

102

107

Means are based on 17-25 animals

Pregnancy performance:

Lower pregnancy frequencies in low and high dose groups compared to control and mid dose groups. These findings are considered incidental as events leading to the establishment of pregnancy should have occurred prior to commencement of treatment.

No indications of an effect of treatment in the frequency of embryo-foetal deaths or on foetal weight.

Foetal abnormalities and variants:

The incidence of major abnormalities did not indicate an effect of treatment. All abnormalities seen at the high dose have been seen previously in the test laboratory.

The incidence of minor visceral abnormalities was similar in all groups.

The incidence of skeletal abnormalities and variants, including the ossification parameters, other than mentioned in the section ¿Details on embryonic and teratogenic effects¿, were essentially similar in all groups.

Table 4: Group incidence of minor foetal abnormalities and variants

Abnormality/Variant

Dose level [mg/kg bw/d]

0

100

500

1000

Incidence of Foetuses (Litters)

Skeletal:

 

 

 

 

Sutural bone

1 (1)

2 (2)

1 (1)

1 (1)

Cervical rib(s)

0

3 (3)

1 (1)

4 (3)

Minimal distortion of rib(s)

0

1 (1)

1 (1)

1 (1)

Misshapen 6th sternebra

1 (1)

1 (1)

1 (1)

0

Asymmetric pelvic girdle

0

0

0

1 (1)

Number of ribs:

 

 

 

 

Reduced 13th rib

0

0

0

1 (1)

13 complete rib(s)

142 (25)

95 (17)

97 (22)

57 (17)

14th vestigial supernummerary rib(s)

12 (9)

14 (8)

37 (15)

55 (15)

Number with minor skeletal abnormality/variant

2 (2)

5 (5)

4 (4)

7 (5)

Total number examined skeletally

154 (25)

109 (17)

134 (22)

113 (19)

Table 5: Group incidence of incomplete ossification parameters

Parameter

Dose level [mg/kg bw/d]

0

100

500

1000

Incidence of Foetuses (Litters)

Incomplete ossification affecting:

 

 

 

 

¿3 skull bones

14 (8)

8 (6)

21 (11)

20 (11)

¿ 4 skull bones

3 (3)

1 (1)

5 (5)

4 (2)

Thoracic vertebral centrum(a)

4 (3)

6 (4)

3 (2)

4 (3)

2nd and 4th metacarpal(s)

1 (1)

0

0

1 (1)

Pubis(es)

1 (1)

1 (1)

0

1 (1)

Ischium

1 (1)

0

0

1 (1)

Cervical vertebral arch(es)

0

0

1 (1)

0

Lumbar vertebral centrum(a)

0

0

0

1 (1)

Sacral vertebral arch(es)

12 (8)

5 (3)

3 (2)

2 (2)

2nd and 4th metatasal(s)

0

0

1 (1)

0

Unossified 5th metacarpal(s)/metatarsal(s)

30 (16)

26 (11)

17 (7)

32 (14)

Number of sternebrae retarded:

 

 

 

0

103 (24)

72 (16)

70 (18)

35 (15)

1

33 (16)

29 (11)

42 (16)

51 (17)

2

17 (9)

8 (5)

20 (9)

20 (11)

3

1 (1)

0

0

2 (1)

>3

0

0

1 (1)

4 (2)

Total number examined

154 (25)

109 (17)

134 (23)

113 (19)

Conclusions:
The source substance 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters caused slight maternal effects only at 1000 mg/kg bw/d indicated by slightly increased body weight gain and food consumption.
Foetal effects were confined to increases in the incidence of vestigial supernumerary ribs at 500 and 1000 mg/kg bw/d and to a slight increase in the incidence of retarded sternebrae at 1000 mg/kg bw/d. Both of these findings were considered to be of a minor nature.
Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 500 mg/kg bw/d. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 1000 mg/kg bw/d.
Executive summary:

A study of developmental toxicity revealed slight maternal effects only at 1000 mg/kg bw/d of the source substance 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters indicated by slightly increased body weight gain and food consumption. Foetal effects were confined to increases in the incidence of vestigial supernumerary ribs at 500 and 1000 mg/kg bw/d and to a slight increase in the incidence of retarded sternebrae at 1000 mg/kg bw/d. Both of these findings were considered to be of a minor nature. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 500 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 1000 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2017 - May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia S.r.l. and supplied by Charles River France
- Age at study initiation: 16-19 weeks (females); at least 25 weeks (males)
- Weight at study initiation: females 2500 g; (males at least 3500 g)
- Fasting period before study: no
- Housing: The rabbits were housed in a limited access facility. During Acclimation period females were group housed, 2 per cage, in polycarbonate/stainless steel cages with perforated NorylTM floor suspended over trays. Males were individually housed in the same type of cages. From allocation, mated females were also individually housed in the same type of cages.
Each cage tray held absorbent paper which was inspected and changed at least 3 times a week.
- Diet: commercially available laboratory rabbit diet ((Mucedola 2 RB 15, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy) ad libitum
- Water: drinking water via water bottles ad libitum
- Acclimation period: An acclimatisation period of at least 30 days was then allowed before the start of pairing, during which time the health status of the animals was assessed by thorough observations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): continous per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2017-04-06 To: 2017-05-23
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): soluble in corn oil, not in water
- Concentration in vehicle: concentrationsof 50, 250 and 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated by the Analytical Chemistry Department at RTC according to RTC SOPs (RTC Study No. A2306).
The purpose of this study was to validate the method suitable to analyse the content of 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters in corn oil. In addition, the stability of the formulations in corn oil was assessed. The chromatograms of the test item showed three main peaks and therefore it was quantified as the sum of these. The validation of the method for the determination of the test item was successful, since linearity, accuracy and precision were all within the limits stated in the Study Protocol. Linearity was successfully assessed in the range from 4.660 to 18.64 µg/mL of the test item, since the correlation coefficient, r,was found to be 0.9999 (acceptability criterion > 0.98). Selectivity was assessed by analysing samples of the diluent and diluent + vehicle. The latter showed a small signal at the retention time of one of the test item peaks, so the calibration standards were prepared in the presence of the vehicle. The accuracy and precision of the method were determined at 10 and 500mg/mL by preparing formulations at these levels and analysing six independent samples from each (two from the top, two from the middle and two from the bottom). At the lower level, accuracy was found to be 93.33%, with a precision, expressed as the percentage coefficient of variation (CV%), of 1.57%, while at the higher level, accuracy was 95.76%, with a precision of 1.39%. These values were within the acceptability criteria outlined in the protocol (accuracy 85 to 115% of the theoretical concentration, precision < 10%). The results at each level were also used as the time-zero content of the formulations for the stability study. The stability of the formulations was assessed after 28 hours and 8 days at room temperature. The results met the acceptability criteria outlined in the protocol (recovery 85 to 115% of the theoretical value, with a homogeneity <10%) at the high level, but not at the low level. Therefore, the formulations were deemed to be stable for 28 hours at room temperature.



Details on mating procedure:
Females were introduced to sexually mature males obtained from the same supplier. Each female remained with the male for at least 1hour after success fulmating had been observed. Each female received 50 I. U. of luteinizing hormone in a marginal ear vein upon completion of the mating procedure. The day successful mating was detected was considered Day 0 post coitum (or gestation Day 0).
Duration of treatment / exposure:
days 6 to 28 post coitum
Frequency of treatment:
1/d
Duration of test:
up to day 28 of gestation
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dose levels have been selected in consultation with the Sponsor based on information from a preliminary, non GLP-compliant study (RTC Study no. Y0090).

Study design of this preliminary study:
A preliminary embryo-foetal developmental toxicity study on LINPLAST 810 P was conducted in the New Zealand rabbit. The effects of LINPLAST 810 P were investigated at the dose levels of 100, 500 and 1000 mg/kg/day when given during gestation. The animals were assigned to 4 groups of 6 mated females.The animals received the test item orally by gavage at constant volume of 2 mL/kg bw from gestation Day 6 up to Day 28 post coitum. Control animals received the vehicle (corn oil) during the same treatment period and dose volume. Clinical signs and body weight were recorded during the in vivo phase. At term, females were caesarean-sectioned on Day 29 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations,early and late intrauterine deaths,live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities.

Results of this preliminary study:
Maternal toxicity: No effects on body weight or clinical signs were recorded during the in vivo phase. Reduced and/soft faeces were observed in all groups regardless the treatment dosing regimen. One low dose animal was sacrificed before the scheduled period, due to the poor health condition,which was considered incidental. At necropsy, the animals sacrificed at term did not show abnormalities that could be considered treatment-related.
Developmental toxicity: The reproductive parameters were comparable between treated and control groups, when considering the number of corpora lutea, the implantation sites or litter size and foetal weight. No major abnormalities at external examination of foetuses were recorded in all treated groups.

In conclusion, based on the results obtained in the study, the dose levels between 100 and 1000 mg/kg/day may be chosen in the subsequent main reproductive toxicity study.

Maternal examinations:
Full records were maintained for all measurements and observations.

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on Days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes,
Food consumption was measured on Days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum starting from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 (injection of Tanax®, Intervet following injection of a sedative/tranquillizer)

The clinical history of the animals was studied and a detailed postmortem examination was conducted (including examination of the external surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin.


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: Yes, only placental remnants visible
- Number of late resorptions: Yes, placental and foetal remnants visible

- Other: number, sex and weight of all live foetuses, number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing), gross evaluation of placentae, uteri or individual uterine horns without visible implantations were immersed in a 20 % solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation
Fetal examinations:
All viable foetuses were euthanised, weighed and examined externally and internally. The thoracic and abdominal cavities were opened and examined and sex was determined. Headsection (medialsuture bones) was performed at necropsy on each foetus selected for skeletal examination and particular attention was paid to the brain ventricles. The head from approximately half of the foetuses (i.e. routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed examination of internal structures of the head: eye, brain, nasal passages and tongue. Foetuses were eviscerated, skinned and fixed in 95% ethanol for subsequent skeletal staining and examination. Skeletal and fixed head examinations were performed in all groups.

Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data. Statistical analyses of non-continuous variables were carried out by means of the Kruskal Wallis test. The criterion of statistical significance was p<0.05.
Indices:
Pre-implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of implantations) x 100/no. of corpora lutea
Post-implantation loss was calculated as a percentage from the formula: (no. of implantations - no. of live young)x100 /no. of implantations
Total implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of live young) x 100/no. of corpora lutea
Sex ratios of the foetuses were calculated as the percentage of males per litter. The number of foetuses affected with structural deviations and the corresponding litter percentage were calculated.
All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.
Clinical signs:
not specified
Description (incidence and severity):
The most recurrent findings were reduced and soft faeces seen in all groups with a similar incidence. Dirty appearance, mucus, red staining and foetuses in cage tray were also recorded in females that aborted or had premature birth. A higher incidence of abortion occurred in the mid - and high dose groups. Damaged ear was found in two high dose females with no toxicological significance.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of seven animals were humanely sacrificed for abortion: one control, two mid-dose and 4 high dose females. The animals aborted between gestation Days 23 and 27. Two animals were found dead, one low dose female on gestation Day26 and one high dose female on gestation Day 22. The low dose female also aborted before its death. One low dose and two mid-dose females had premature birth on gestation Day 27 and on gestation Day 29, before necropsy, respectively. One female of each group was found not pregnant; in the control, low, mid-and high dose groups respectively). The number of females with live foetuses on gestation Day 29 was: 18 in the control group, 17 in the low dose group,15 in the mid-dose group and 14 in the high dose group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The pregnancy losses of the animals that aborted or had premature birth were preceded by constant trend of decreased body weight compared to the value at the start of treatment (Day 6 post coitum).


Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
A general trend of reduction in food consumption was noted through the study in all groups. This reduction was more evident in females that aborted or had premature birth.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were found in terminal body weight,uterus weight and absolute weight gain between groups.
Gross pathological findings:
no effects observed
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
A higher incidence of abortion occurred in the mid-and high dose groups.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Loss of pregnancy occurred in all groups although an increased incidence was found in the mid-and high dose females. The animals that aborted or had premature birth, behind the presence of foetuses in the cage tray, showed a similar pattern of findings like red staining and mucus in the cage tray, dirty urogenital region and a marked trend of body weight loss along with reduction in food consumption. At daily clinical observations, reduced and/or soft faeces were similarly recorded in all groups and likely due to the disturbance of the gastrointestinal tract. However, there were no changes in body weight or food consumption data through the study. The gross observations, in particular for animals that aborted, revealed abnormal content, size and colour of the caecum. Additional changes were seen in the stomach, jejunum and ileum. At final sacrifice, abnormal content of the caecum was still present with a dose-related trend.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
number of abortions
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was a similar incidence of small foetuses (body weight below 35 g) between groups. Dead foetuses (n=5) were recorded in one low dose female and the other foetuses of the same litter were all small.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No anomalies were detected in all groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A similar incidence of the most frequent findings classified as variants or anomalies, were recorded in all groups and included, for example, the supernumerary rib(13th), the incomplete or no ossification of 5th and 6th sternebrae, no ossification of humerus, femur and tibia heads and the bilateral insertion at the 2nd sacral vertebrae of the pelvic girdle. In addition, the number of incomplete and/or no ossification of several bones such as proximal or distal forepaw/hindpaw phalanges including the 1st proximal phalanx, pubis, no ossification of trochlea and also a stragalus, the enlarged anterior and posterior fontanelle of the skull, were more frequently found in small foetuses, with body weight less than 35g, indicating that the extent of ossification may be related to a foetal growth retardation. Two major anomalies were found in the control and high dose group, related to the absence of the articulation point in the pelvic girdle and one thoracic emivertebrae (8th). These malformations occurred in the control and high dose groups and therefore considered incidental findings. Other anomalies included fused, bipartite and/or asymmetrically shaped sternebrae and outward rotation of the forepaw(s) occasionally found in the control and treated groups.

Visceral malformations:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Fixed head examination did not reveal differences between groups. A similar pattern of findings was recorded in all groups and generally included slight or moderate enlarged ventricles and folded retina.
Details on embryotoxic / teratogenic effects:
No toxic effects were seen on the developmental outcome. The foetuses at term (gestation Day29) did not show substantial differences between control and treated groups including external/internal examination, skeletal and fixed head data.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Clinical signs

 
 Group

a (b)

100 mg/kg bw/d 

a (b)

500 mg/kg bw/d 

a (b)

1000 mg/kg bw/d 

a (b)

Staining (appearance)  0 (0%) 1 (5 %)  0 (0 %)  0 (0 %)
Hairloss (apperance) 2 (10 %)  0 (0 %)  1 (5 %)  0 (0 %)

Red staining on cage tray

(Urogenital)

 1 (5 %)  1 (5 %)  4 (20 %)  3 (15 %)
 Foetuses on cage tray (Urogenital)  1 (5 %)  2 (10 %)  4 (20 %)  4 (20%)
 Mucus on cage tray (Urogenital)  0 (0%)  1 (5 %)  0 (0%)  0 (0 %)
 Dirty (Urogenital)  0 (0 %) 1 (5 %)   0 (0 %)  1 (5 %)
 Soft faeces (Digestive System)  1 (5 %)  1 (5 %)  1 (5 %)  1 (5 %)
Reduced faeces (Digestive System)   16 (80 %)  8 (40 %) 10 (50 %)  11 (55 %) 
 Damaged ear (Eye-Ear-Mouth)  0 (0 %) 0 (0 %)  0 (0 %)  2 (10 %) 

a = number of animals affected ( b = percentage of animals with observation during interval)

Table 2: Mortality and Fate of females - group incidence
Negative control group Low dose Medium dose High dose
0 mg/kg bw/d 100 mg/kg bw/d 500 mg/kg bw/d 1000 mg/kg bw/d
Initial group size 20 20 20 20
Not pregnant 1 1 1 1
Premature birth 0 1 2 0
Human kill (abortion) 1 0 2 4
Found dead 0 1 0 1
With live foetuses on day 29 post coitum 18 17 15 14
Table 3: Body weight of females (kg) - group mean data
Day of phase
0G 3 6D 9 12 15 18 21 23 26 29
Negative control (0 mg/kgbw/d) (n) 19 19 19 19 19 19 19 19 19 19 18
mean 3.902 4.015 4.059 3.998 4.009 4.067 4.039 4.020 4.017 4.014 4.029
SD 0.349 0.332 0.331 0.344 0.278 0.305 0.284 0.292 0.270 0.270 0.300
Low dose (100 mg/kg bw/d) (n) 19 19 19 19 19 19 19 19 19 18 17
mean 3.870 3.946 4.008 3.937 3.969 4.036 4.003 3.990 3.970 4.001 3.968
SD 0.441 0.421 0.395 0.386 0.351 0.341 0.302 0.246 0.247 0.199 0.187
Medium dose (500 mg bw/d)) (n) 19 19 19 19 19 19 19 19 19 19 17
mean 3.883 3.959 4.029 3.953 3.956 4.018 3.992 3.996 3.989 3.966 3.960
SD 0.400 0.388 0.374 0.352 0.315 0.291 0.280 0.278 0.290 0.337 0.346
High dose (1000 mg bw/d) (n) 19 19 19 19 19 19 19 19 18 15 14
mean 3.949 4.027 4.079 4.053 4.047 4.037 3.997 3.986 4.007 4.084 4.095
SD 0.307 0.312 0.312 0.313 0.311 0.284 0.296 0.317 0.350 0.332 0.289
G= Gestation phase;D= Dosing/gestation phase; * = mean value of group is significantly different from control at p<0.05 (Dunnett's test)
Table 4: Body weight gain per dayO(g) of females - group mean data
Day of phase
3G 6D 9 12 15 18 21 23 26 29
Negative control (0 mg/kg bw/d) (n) 19 19 19 19 19 19 19 19 19 18
mean 37.6719 14.7368 -20.496 3.8193 19.2491 -3.8807 -6.5334 -1.1474 -1.0737 -5.6334
SD 30.9206 24.8099 34.9321 35.5196 38.3050 28.9602 28.7688 45.8125 27.8992 34.3498
Low dose (100 mg/kg bw/d) (n) 19 19 19 19 19 19 19 19 18 17
mean 25.500 20.3947 -23.542 10.4860 22.4368 -11.028 -4.0789 -10.186 0.5611 -9.5686
SD 27.1239 28.7108 31.2713 34.085 30.9303 32.5985 30.7487 29.9979 29.9600 44.0406
Medium dose (500 mg/kg bw/d) (n) 19 19 19 19 19 19 19 19 19 17
mean 25.3737 23.1386 -25.133 0.7702 20.7263 -8.6544 1.3018 -3.1684 -7.6965 -25.864
SD 20 14.2782 32.1613 36.0484 33.8138 37.3572 25.2199 29.7963 35.4355 54.5322
High dose (1000 mg/kg bw/d)) (n) 19 19 19 19 19 19 19 18 15 14
mean 25.8631 17.3386 -8.5702 -2.0842 -3.2965 -13.433 -3.6263 1.3584 -7.5022 -12.961
SD 32.6529 11.8222 24.5874 37.3870 43.4562 43.8186 32.7626 56.5149 55.7807 29.9024
O= mean daily body weight gain over the previous period;G= Gestation phase;D= Dosing/gestation phase
* = mean value of group is significantly different from control at p<0.05
** = mean value of group is significantly different from control at p<0.01
Statistical analysis: Dunnett's test (group variances are homogeneous); $ = Modified t-test (group variances are inhomogeneous)
Table 5: Food consumptionO(g/animals/day) of females - group mean data
Day of phase
3G 6D 9 12 15 18 21 23 26 29
Negative control (0 mg/kg bw/d) (n) 19 19 19 19 19 19 19 19 19 18
mean 178.43 187.80 146.19 142.09 135.41 124.01 128.25 138.98 105.87 81.38
SD 20.02 23.19 36.34 37.47 47.08 68.74 51.98 55.13 55.26 35.75
Low dose (100 mg/kg bw/d) (n) 19 19 19 19 19 19 19 19 19 16
mean 167.90 188.81 131.18 127.79 134.39 110.90 133.68 125.22 113.43 85.75
SD 29.39 26.80 39.93 46.38 38.37 49.46 42.82 63.23 41.50 43.32
Medium dose (500 mg/kg bw/d) (n) 19 19 19 19 19 19 19 19 19 17
mean 173.77 189.16 127.13 128.51 130.28 124.21 133.66 118.81 112.99 72.06
SD 22.93 23.61 40.31 37.94 38.01 36.23 41.96 56.91 40.11 30.32
High dose (1000 mg/ kg bw/d) (n) 19 19 19 19 19 19 19 18 15 14
mean 175.17 187.35 142.48 140.74 136.27 125.41 123.42 112.51 119.32 88
SD 23.34 29.67 41.74 46.68 45.28 37.31 41.72 56.24 45.53 29.45
O= food consumed over the previous period starting from allocation;G= Gestation phase;D= Dosing/gestation phase
* = mean value of group is significantly different from control at p<0.05
** = mean value of group is significantly different from control at p<0.01
Statistical analysis: Dunnett's test (group variances are homogeneous); $ = Modified t-test (group variances are inhomogeneous)
Table 6: Terminal body weight, uterus weight and absolute weight gain of females with live foetuses - group mean data
Terminal body weight (kg) Gravid uterus weight (g) Absolute weight gain# (g)
Negative control (0 mg/kg bw/d) (n) 18 18 18
mean 3.98 494.94 -410.73
SD 0.37 75.45 346.29
Low dose (100 mg/kg bw/d) (n) 17 17 17
mean 3.90 477.87 -377.30
SD 0.29 86.63 353.31
Medium dose (500 mg/kg bw/d) (n) 15 15 15
mean 4.00 461.39 -278.34
SD 0.35 106.55 216.54
High dose (1000 mg/kg bw/d) (n) 14 14 14
mean 4.02 479.11 -384.19
SD 0.40 155.03 327.40
# = body weight at necropsy minus gravid uterine wt., minus body wt. at Day 0 of pregnancy
* = statistically significantly different from control group value at p<0.05
Table 7: Litter data and sex ratios - group mean data
Uterine deaths Viable young % Implantation loss (%)
Corpora Lutea Implantations Early Late Total Total M F Males Pre Post Total Litter weight (g) Mean foetal weight (g)
Negative control (0) (n) 18 18 18 18 18 18 18 18 18 18 18 18 18 18
mean 9.72 9.61 0.17 0.11 0.28 9.33 4.39 4.94 47.35 1.06 2.63 3.70 339.4 37.29
SD 2.11 2.09 0.71 0.32 0.75 2.09 1.69 1.83 14.69 3.09 7.38 7.63 62.83 6.41
Low dose (100 mg/kg bw/d) (n) 17 17 17 17 17 17 17 17 17 17 17 17 17 17
mean 9.53 9.18 0.00 0.29 0.59 8.59 4.12 4.76 46.65 3.88 5.18 9.06 311.5 36.35
SD 2.10 2.16 0.00 0.59 1.70 2.18 1.62 1.71 14.77 7.24 15.39 15.71 80.15 6.50
Medium dose (500 mg/kg bw/d) (n) 15 15 15 15 15 15 15 15 15 15 15 15 15 15
mean 8.67 8.60 0.53 0.07 0.60 8.00 4.27 3.73 52.79 0.55 7.39 7.74 308.6 39.19
SD 1.99 1.88 1.13 0.26 1.24 1.96 1.79 1.49 15.47 2.14 13.73 14.57 75.35 4.72
High dose (1000 mg/kg bw/d) (n) 14 14 14 14 14 14 14 14 14 14 14 14 14 14
mean 9.21 8.71 0.07 0.00 0.07 8.64 3.86 4.79 45.40 5.72 2.38 7.50 330.3 39.61
SD 2.72 2.55 0.27 0.00 0.27 2.73 1.66 2.15 13.85 8.49 8.90 13.81 107.0 8.29
* = statistically significantly different from control group value at p<0.05
Table 8: Macroscopic observations - Unscheduled death - group incidence
Females
Negative control group Low dose Medium dose High dose
0 mg/kg bw/d 100 mg/kg bw/d 500 mg/kg bw/d 1000 mg bw/d
Number in group 1 2 2 5
Abdominal cavity
Abnormal contents 0 0 0 1
Caecum
Abnormal colour 0 0 0 1
Abnormal size 0 0 0 1
Abnormal contents 1 0 2 3
Gall bladder
Abnormal size 0 1 0 1
Heart
Abnormal area(s) 0 0 0 1
Ileum
Abnormal colour 0 0 0 1
Jejenum
Abnormal colour 0 0 0 1
Abnormal contents 0 0 0 1
Liver
Abnormal colour 1 2 0 1
Lungs
Abnormal area(s) 0 1 0 1
Abnormal colour 0 1 0 0
Skin
Staining 1 1 1 3
Stomach
Abnormal area(s) 0 0 0 1
Table 9: Macroscopic observations - final sacrifice - group incidence
Negative control group Low dose Medium dose High dose
0 mg/kg bw/d 100 mg/kg bw/d 500 mg/kg bw/d 1000 mg/kg bw/d
Number in group
Caecum
Abnormal contents 0 1 2 2
Ears
Abnormal area(s) 0 0 0 2
Forelimbs
Hairloss 2 0 1 0
Liver 
Abnormal colour 0 0 1 0
Skin 
Staining  0 1 1 0
Uterus
Not pregant 1 1 1 1
Whole animal
No abnormalities detected 15 15 14 11
Table 10: External and internal examination of foetuses - group incidence
No. foetuses No. litters
Group Organ Cat Observation(s) Observed Affected % Observed Affected %
Negative control (0 mg/kg bw/d) Whole foetus No abnormalities detected 168 105 62.50 18 - -
AN Small 168 63 37.50 18 14 77.78
Low dose (100 mg/kg bw/d) Whole foetus No abnormalities detected 151 99 65.56 17 - -
Dead foetus 151 5 3.31 17 1 5.88
AN Small 151 47 31.13 17 11 64.71
Medium dose (500 mg bw/d) Whole foetus No abnormalities detected 120 93 77.50 15 - -
AN Small 120 27 22.50 15 9 60.00
High dose (1000 mg bw/d) Whole foetus No abnormalities detected 121 87 71.90 14 - -
AN Small 121 34 28.10 14 6 42.86
Table 10: Skleletal examination of foetuses - group incidence
No. foetuses No. dams
Group Organ Cat Observation(s) Observed Affected % Observed Affected %
Negative control (0 mg/kg bw/d) Cervical vertebrae AN Cervical rib(s) 168 1 0.60 18 1 5.56
Forelimb(s) AN Humerus head no ossification 168 81 48.21 18 16 88.89
Forlelimb(s) AN Humerus trochlea no ossification 168 23 13.69 18 7 38.89
Forepaw(s) AN Distal phalanx no ossification 1st 168 1 0.60 18 1 5.56
Forepaw(s) AN Distal phalanx incomplete ossification 1st 168 2 1.19 18 1 5.56
Forepaw(s) AN Abnormal shape 168 1 0.60 18 1 5.56
Forepaw(s) AN One or more distal phalange(s) incomplete ossification 168 22 13.10 18 13 72.22
Forepaw(s) AN One or more distal phalange(s) no ossification 168 28 16.67 18 12 66.67
Forepaw(s) AN Proximal phalanx incomplete ossification 1st 168 1 0.60 18 1 5.56
Forepaw(s) AN Proximal phalanx no ossification 1st 168 6 3.57 18 3 16.67
Hindlimb(s) AN Femur head no ossificatioin 168 65 38.69 18 15 83.33
Hindlimb(s) VA Tibia head no ossification 168 112 66.67 18 18 100
Hindpaw(s) AN Astragalus no ossification 168 2 1.19 18 2 11.11
Hindpaw(s) VA One or more distal phalange(s) no ossification 168 2 1.19 18 2 11.11
Pelvic girdle AN Articulation point reduced 168 5 2.98 18 4 22.22
Pelvic girdle AN Additioinal article point 168 27 16.07 18 8 44.44
Pelvic girdle MA Articulation point absent 168 3 1.79 18 3 16.67
Pelvic girdle VA Unilateral insertion 2nd sacral vertebrae 168 5 2.98 18 4 22.22
Pelvic girdle VA Bilateral insertion 2nd sacral vertebrae 168 26 15.48 18 9 50
Ribs AN Floating 13th 168 17 10.12 18 9 50
Ribs VA Unilateral 13th 168 29 17.26 18 14 77.78
Ribs VA 13 pairs 168 78 46.43 18 18 100
Ribs VA Rudimentary 13th 168 2 1.19 18 2 11.11
Ribs VA Short 13th 168 53 31.55 18 15 83.33
Skull AN Hyoid body incomplete ossification 168 9 5.36 18 6 33.33
Skull AN Hyoid body no ossification 168 8 4.76 18 4 22.22
Skull VA Hyoid horn incomplete ossification 168 1 0.60 18 1 5.56
Skull VA Anterior fontanelle enlarged 168 18 10.71 18 8 44.44
Sternebrae AN Rudimentary 6th 168 1 0.60 18 1 5.56
Sternebrae AN One or more fused 168 1 0.60 18 1 5.56
Sternebrae AN No ossification 6th 168 10 5.95 18 7 38.89
Sternebrae AN Asymmetrical ossification 6th 168 1 0.60 18 1 5.56
Sternebrae AN Bipartite 6th 168 3 1.79 18 1 5.56
Sternebrae VA Incomplete ossification 5th 168 45 26.79 18 18 100
Sternebrae VA No ossification 5th 168 35 20.83 18 10 55.56
Sternebrae VA Incomplete ossification 6th 168 12 7.14 18 6 33.33
Sternebrae VA Rudimentary 5th 168 7 4.17 18 4 22.22
Whole foetus no No abnormalities detected 168 5 2.98 18 4 22.22
Low dose (100 mg/kg bw/d) Caudal Vertebrae AN Less tthan 12 146 1 0.68 17 1 5.88
Forelimb(s) AN Humerus head no ossification 146 69 47.26 17 13 76.47
Forelimb(s) AN Humerus trochlea no ossification 146 14 9.59 17 5 29.41
Forepaw(s) AN One or more distal phalange(s) no ossification 146 20 13.70 17 7 41.18
Forepaw(s) AN Distal phalanx incomplete ossification 1st 146 2 1.37 17 2 11.76
Forepaw(s) AN Abnormal shape 146 3 2.05 17 3 17.65
Forepaw(s) AN Proximal phalanx no ossification 1st 146 9 6.16 17 5 29.41
Forepaw(s) AN One or more distal phalange(s) incompomplete ossification 146 11 7.53 17 8 47.06
Forepaw(s) AN Distal phalanx no ossification 1st 146 1 0.68 17 1 5.88
Forepaw(s) AN Proximal phalanx incomplete ossification 1st 146 6 4.11 17 5 29.41
Hindlimb(s) AN Femur head no ossificatioin 146 52 35.62 17 13 76.47
Hindlimb(s) VA Tibia head no ossification 146 94 64.38 17 16 94.12
Hindpaw(s) AN Astragalus no ossification 146 3 2.05 17 2 11.76
Hindpaw(s) AN Astragalus incomplete ossification 146 3 2.05 17 2 11.76
Hindpaw(s) VA One or more proximal phalange(s) incomplete ossification 146 2 1.37 17 1 5.88
Hindpaw(s) VA One or more proximal phalange(s) no ossification 146 1 0.68 17 1 5.88
Hindpaw(s) VA One or more distal phalange(s) no ossification 146 3 2.05 17 2 11.76
Pelvic girdle AN Articulation point reduced 146 3 2.05 17 2 11.76
Pelvic girdle AN Additional articulation point 146 49 33.56 17 16 94.12
Pelvic girdle AN Pubis no ossification 146 1 0.68 17 1 5.88
Pelvic girdle AN Pubis incomplete ossification 146 4 2.74 17 2 11.76
Pelvic girdle VA Unilateral insertion 2nd sacral vertebrae 146 2 1.37 17 2 11.76
Pelvic girdle VA Bilateral insertion 2nd sacral vertebrae 146 56 38.36 17 14 82.35
Ribs AN Floating 13th 146 17 11.64 17 10 58.82
Ribs VA Short 13th 146 57 39.04 17 16 94.12
Ribs VA Rudimentary 13th 146 1 0.68 17 1 5.88
Ribs VA Unilateral 13th 146 28 19.18 17 14 82.35
Ribs VA 13 pairs 146 88 60.27 17 15 88.24
Skull AN Hyoid body incomplete ossification 146 10 6.85 17 5 29.41
Skull AN Frontal fissure 146 1 0.68 17 1 5.88
Skull AN Hyoid body no ossification 146 3 2.05 17 3 17.65
Skull VA Anterior fontanelle enlarged 146 13 8.90 17 7 41.18
Skull VA Posterior fontanelle enlarged  146 2 1.37 17 1 5.88
Sternebrae AN Bipartite 6th  146 1 0.68 17 1 5.88
Sternebrae AN No ossification 6th 146 10 6.85 17 6 35.29
Sternebrae AN Rudimentary 6th  146 3 2.05 17 3 17.65
Sternebrae AN Asymetrical ossification 6th  146 4 2.74 17 4 23.53
Sternebrae AN Asymetrical ossification 5th  146 2 1.37 17 2 11.76
Sternebrae AN One or more asymmetrical ossification 146 4 2.74 17 4 23.53
Sternebrae AN Bipartite 5th 146 1 0.68 17 1 5.88
Sternebrae VA One or more incomplete ossification 146 1 0.68 17 1 5.88
Sternebrae VA No ossification 5th 146 17 11.64 17 6 35.29
Sternebrae VA Incomplete ossification 5th 146 44 30.14 17 15 88.24
Sternebrae VA Incomplete ossification 6th  146 14 9.59 17 6 35.29
Sternebrae VA Rudimentary 5th 146 12 8.22 17 9 52.94
Whole foetus no No abnormalities detected 146 5 3.42 17 5 29.41
Medium dose (500 mg/kg bw/d) Forelimb(s) AN Humerus head no ossification 120 58 48.33 15 14 93.33
Forelimb(s) AN Humerus trochlea no ossification 120 2 1.67 15 2 13.33
Forepaw(s) AN One or more distal phalange(s) no ossification 120 23 19.17 15 9 60.00
Forepaw(s) AN One or more distal phalange(s) incompomplete ossification 120 9 7.50 15 7 46.67
Hindlimb(s) AN Femur head no ossificatioin 120 32 26.67 15 11 73.33
Hindlimb(s) VA Tibia head no ossification 120 86 71.67 15 15 100
Lumbar vertebrae AN One or more centra bipartite  120 1 0.83 15 1 6.67
Lumbar vertebrae AN One or more arches no ossification 120 1 0.83 15 1 6.67
Lumbar vertebrae AN One or more centra incomplete ossification 120 1 0.83 15 1 6.67
Lumbar vertebrae AN One or more centra fused 120 1 0.83 15 1 6.67
Lumbar vertebrae VA Lumbar spinous process present 4th 120 1 0.83 15 1 6.67
Pelvic girdle AN Additional articulation point 120 41 34.17 15 13 86.67
Pelvic girdle AN Articulation point reduced 120 4 3.33 15 3 20.00
Pelvic girdle VA Unilateral insertion 2nd sacral vertebrae 120 2 1.67 15 2 13.33
Pelvic girdle VA Bilateral insertion 2nd sacral vertebrae 120 20 16.67 15 12 80.00
Ribs AN Floating 13th 120 13 10.83 15 7 46.67
Ribs VA 13 pairs 120 63 52.50 15 14 93.33
Ribs VA Short 13th 120 49 40.83 15 15 100.00
Ribs VA Unilateral 13th 120 16 13.33 15 9 60.00
Skull AN Hyoid horn bent 120 1 0.83 15 1 6.67
Skull AN Hyoid body no ossification 120 1 0.83 15 1 6.67
Skull AN Frontal fissure 120 1 0.83 15 1 6.67
Skull VA Anterior fontanelle enlarged 120 4 3.33 15 4 26.67
Sternebrae AN Bipartite 5th 120 1 0.83 15 1 6.67
Sternebrae AN Rudimentary 6th 120 5 4.17 15 4 26.67
Sternebrae AN One or more asymmetrical ossification 120 10 8.33 15 9 60.00
Sternebrae AN No ossification 6th  120 6 5.00 15 3 20.00
Sternebrae AN Bipartite 6th 120 1 0.83 15 1 6.67
Sternebrae VA Rudimentary 5th  120 10 8.33 15 7 46.67
Sternebrae VA No ossification 5th 120 21 17.50 15 10 66.67
Sternebrae VA Incomplete ossification 5th 120 46 38.33 15 15 100.00
Sternebrae VA Incomplete ossification 6th 120 14 11.67 15 8 53.33
Whole foetus No No abnormalities detected 120 1 0.83 15 1 6.67
High dose (1000 mg/kg bw/d) Forelimb(s) AN Humerus head no ossification 121 44 36.36 14 9 64.29
Forelimb(s) AN Humerus trochlea no ossification 121 10 8.26 14 3 21.43
Forepaw(s) AN One or more distal phalange(s) incompomplete ossification 121 5 4.13 14 3 21.43
Forepaw(s) AN One or more distal phalange(s) no ossification 121 7 5.79 14 4 28.57
Forepaw(s) AN Abnormal shape 121 1 0.83 14 1 7.14
Forepaw(s) AN Proximal phalanx no ossification 1st 121 10 8.26 14 4 28.57
Forepaw(s) AN Proximal phalanx incomplete ossification 1st 121 4 3.31 14 2 14.29
Hindlimb(s) AN Femur head no ossificatioin 121 39 32.23 14 8 57.14
Hindlimb(s) VA Tibia head no ossification 121 57 47.11 14 9 64.29
Hindpaw(s) AN Astralagus incomplete ossification 121 6 4.96 14 1 7.14
Hindpaw(s) AN Astralagus no ossification 121 2 1.65 14 1 7.14
Lumbar vertebrae VA Lumbar spinous process present 4th 121 3 2.48 14 2 14.29
Pelvic girdle AN Articulation point reduced 121 2 1.65 14 2 14,29
Pelvic girdle AN Pubis incomplete ossification 121 7 5.79 14 1 7.14
Pelvic girdle AN Addtional articulation point 121 57 47.11 14 14 100.00
Pelvic girdle AN Pubis no ossification 121 1 0.83 14 1 7.14
Pelvic girdle MA Articulation point absent 121 1 0.83 14 1 7.14
Pelvic girdle VA Bilateral insertion 2nd sacral vertebrae 121 32 26.45 14 11 78.57
Ribs AN Floating 13th 121 17 14.05 14 8 57.14
Ribs VA 13 pairs 121 80 66.12 14 14 100.00
Ribs VA Unilateral 13th 121 14 11.57 14 8 57.14
Ribs VA Short 13th 121 64 52.89 14 14 100.00
Ribs VA Rudimentary 13th 121 1 0.83 14 1 7.14
Skull AN Hyoid horn bent 121 1 0.83 14 1 7.14
Skull AN Frontal fissure 121 3 2.48 14 2 14.29
Skull VA Anterior fontanelle enlarged 121 16 13.22 14 5 35.71
Skull VA Posterior fontanelle enlarged  121 5 4.13 14 1 7.14
Sternebrae AN One or more incomplete ossification 121 1 0.83 14 1 7.14
Sternebrae AN Bipartite 6th 121 1 0.83 14 1 7.14
Sternebrae AN One or more bipartite 121 1 0.83 14 1 7.14
Sternebrae AN One or more abnormal shaped 121 2 1.65 14 2 14.29
Sternebrae AN No ossification 6th 121 7 5.79 14 2 14.29
Sternebrae AN Bipartite 5th 121 2 1.65 14 2 14.29
Sternebrae AN Asymmetrical ossification 6th  121 2 1.65 14 1 7.14
Sternebrae AN One or more asymmetrical ossification  121 6 4.96 14 4 28.57
Sternebrae AN Rudimentary 6th 121 4 3.31 14 3 21.43
Sternebrae AN One or more fused 121 2 1.65 14 2 14.29
Sternebrae VA Rudimentary 5th 121 15 12.40 14 10 71.43
Sternebrae VA No ossification 5th 121 23 19.01 14 11 78.57
Sternebrae VA Incomplete ossification 6th 121 8 6.61 14 7 50.00
Sternebrae VA Incomplete ossification 5th 121 38 31.40 14 12 85.71
Thoracic vertebrae MA One or more hemivertebrae 121 1 0.83 14 1 7.14
Whole foetus no No abnormalities detected 121 1 0.83 14 1 7.14
Conclusions:
It can be concluded that maternal toxicity was observed in the mid-and high dose groups due to a higher incidence of loss of pregnancy. The animals likely had disturbance of gastro intestinal tract more evident in these groups. In such condition the animals reduced food intake and had body weight loss. The final result of this discomfort was the abortion/premature birth. No toxic effects were seen on the developmental outcome. The foetuses at term (gestation Day29) did not show substantial differences between control and treated groups including external/internal examination, skeletal and fixed head data. Therefore, it can be confirmed that the substance, based on our experimental condition, did not affect the normal development of foetuses. Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity is 100 mg/kg/day and 1000 mg/kg/day for developmental toxicity.
Executive summary:

The aim of this study was to investigate the toxicity of 1,2 -Benzenedicarboxylicacid, di-C8 -10alkylesters in New Zealand rabbits during pregnancy and embryo-foetal development, when given by oral gavage. In-house mated females received the test item, formulated in corn oil, at 100, 500 and 1000 mg/kg/day from Day 6 through Day 28 post coitum at constant volume of 2mL/kg body weight.

Maternal toxicity: Loss of pregnancy occurred in all groups although an increased incidence was found in the mid-and high dose females. The animals that aborted or had premature birth, behind the presence of foetuses in the cage tray, showed a similar pattern of findings like red staining and mucus in the cage tray, dirty urogenital region and a marked trend of body weight loss along with reduction in food consumption. At daily clinical observations, reduced and/or soft faeces were similarly recorded in all groups and likely due to the disturbance of the gastrointestinal tract. However, there were no changes in body weight or food consumption data through the study. The gross observations, in particular for animals that aborted, revealed abnormal content, size and colour of the caecum. Additional changes were seen in the stomach, jejunum and ileum. At final sacrifice, abnormal content of the caecum was still present with a dose-related trend.

Developmental toxicity: The overall pregnant rates were similar in all groups and close to 100%. The intrauterine and ovaries examinations did not show findings of toxicological relevance. Comparable values of implantation loss, litter and mean foetal weights were found between groups. Sex ratios were also unaffected by treatment. The skeletal and fixed head examinations did not reveal treatment-related findings.

It can be concluded that maternal toxicity was observed in the mid-and high dose groups due to a higher incidence of loss of pregnancy. The animals likely had disturbance of gastrointestinal tract more evident in these groups. In such condition the animals reduced food intake and had body weight loss. The final result of this discomfort was the abortion/premature birth. No toxic effects were seen on the developmental outcome. The foetuses at term (gestation Day 29) did not show substantial differences between control and treated groups including external/internal examination, skeletal and fixed head data. Therefore, it can be confirmed that the test substance, based on our experimental condition, did not affect the normal development of foetuses. Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity is 100 mg/kg/day and 1000 mg/kg/day for developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Klimisch 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The developmental toxicity accordingt to OECD 414 of the target substance 1,2 -Benzenedicarboxylicacid, di-C8 -10alkylesters was conducted in New Zealand rabbits during pregnancy and embryo-foetal development, when given by oral gavage. In-house mated females received the target substance, formulated in corn oil, at 100, 500 and 1000 mg/kg/day from Day 6 through Day 28 post coitum at constant volume of 2mL/kg body weight.

Maternal toxicity: Loss of pregnancy occurred in all groups although an increased incidence was found in the mid-and high dose females. The animals that aborted or had premature birth, behind the presence of foetuses in the cage tray, showed a similar pattern of findings like red staining and mucus in the cage tray, dirty urogenital region and a marked trend of body weight loss along with reduction in food consumption. At daily clinical observations, reduced and/or soft faeces were similarly recorded in all groups and likely due to the disturbance of the gastrointestinal tract. However, there were no changes in body weight or food consumption data through the study. The gross observations, in particular for animals that aborted, revealed abnormal content, size and colour of the caecum. Additional changes were seen in the stomach, jejunum and ileum. At final sacrifice, abnormal content of the caecum was still present with a dose-related trend.

Developmental toxicity: The overall pregnant rates were similar in all groups and close to 100%. The intrauterine and ovaries examinations did not show findings of toxicological relevance. Comparable values of implantation loss, litter and mean foetal weights were found between groups. Sex ratios were also unaffected by treatment. The skeletal and fixed head examinations did not reveal treatment-related findings.

It can be concluded that maternal toxicity was observed in the mid-and high dose groups due to a higher incidence of loss of pregnancy. The animals likely had disturbance of gastrointestinal tract more evident in these groups. In such condition the animals reduced food intake and had body weight loss. The final result of this discomfort was the abortion/premature birth. No toxic effects were seen on the developmental outcome. The foetuses at term (gestation Day 29) did not show substantial differences between control and treated groups including external/internal examination, skeletal and fixed head data. Therefore, it can be confirmed that the test substance, based on our experimental condition, did not affect the normal development of foetuses. Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity is 100 mg/kg/day and 1000 mg/kg/day for developmental toxicity in rabbits.

A study of developmental toxicity has been undertaken in rats on the source substance, phthalic acid di-n-hexyl, di-n-octyl, n-decyl ester which has C-chain lengths of C6, C10 compared to 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters C8 and C10. In this study slight maternal effects were observed at 1000 mg/kg bw/day indicated by slightly increased body weight gain and food consumption. Foetal effects were confined to increases in the incidence of vestigial supernumerary ribs at 500 and 1000 mg/kg bw/day and to a slight increase in the incidence of retarded sternebrae at 1000 mg/kg bw/day. Both of these findings were considered to be of a minor nature. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 500 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 1000 mg/kg bw/day.

On the basis of all evaluated data, the similarity of the read-across substances is justified on basis of similar composition, structural similarity, the physico-chemical properties, toxicokinetics and toxicological profiles and supported by various QSAR methods. Therefore the read-across approach is used to fill data gaps. There is convincing evidence that the substances have an overall common category profile in regard to this endpoints. The substances have the same basic structure (diester of 1,2-Benzenedicarboxylic acid), with almost identical and overlapping alkyl chains. All of the substances (the target substance 1,2-Benzenedicarboxylic acid, C8 -10 -alkyl esters as well as the read-across substance) are esters. The target substance is a mixture of different isomers, with ca. 21 % dioctyl phthalate and ca. 28 % didecyl phthalate and ca. 48 % decyl octyl phthalate. Read-across substance 1,2-Benzenedicarboxylic acid, C6-10-alkyl esters (CAS-number 68515-51-5), is also a mixture of different isomers, with ca. 33 % dioctyl phthalate and ca. 18 % hexyl octyl phthalate and ca. 30 % decyl octyl phthalate and ca. 7.5 % didecyl phthalate. Therefore, the isomers of the target substance are also constituents of the read-across substance 1. Therefore it is likely that the result of the study is valid for the test substance.

A similar study of developmental toxicity has been undertaken on a structurally related substance,1,2 benzene dicarboxylic acid , di C9-C11 alkyl esters which has C-chain lengths of C9, C11 compared to 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters C8 and C10. In this study no clear adverse effects on pregnancy of the female rat or on embryofoetal survival and development were apparent at dose levels up to 1000 mg/kg bw/day.

Toxicity to reproduction: other studies

Additional information

The structurally related substance Di-n-octyl-phthalate (DnOP) did not induce oestrogenic responses in vivo in a uterotrophic and vaginal cornification assay using immature and mature ovariectomised rats. DnOP was negative for oestrogenic activity in a recombinant yeast assay. The chemical was also not a competitive agonist at the oestrogen receptor in an in vitro competitive ligand-binding assay and did not induce oestrogen receptormediated gene expression in MCF-7 cells. 40 Phthalate-induced germ-cell detachment was examined in vitro in co-cultures of Sertoli and germ cells isolated from pubertal rats. Mono-n-octyl phthalate induced marked germ-cell detachment and disruption of the Sertoli-cell monolayer at 10−4 M, the magnitude of which exceeded that of the other eight monoesters tested. DnOP (up to 10−3 M) showed no detectable binding to oestrogen receptor alpha or beta in vitro. DnOP demonstrated neither agonistic nor antagonistic activity in in vitro reporter gene assays with CHO-K1 cells transfected with expression vectors for human oestrogen receptor alpha, and no anti-androgenic effects were reported (cited in NICNAS, 2015).

NICNAS, 2015; Existing Chemical Hazard Assessment Report, di-n-octyl phthalate, Australian Government of Health and Ageing NICNAS

Justification for classification or non-classification

Based on these results the target substance does not require classification according to Regulation (EC) No 1272/2008.