Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: Read-across from experimental results on the substance 6-tert-Butyl-2,4-xylenol: Test according to OECD guideline 422. GLP study.

NOAELs for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively.  

Data waiving: In accordance with column 1 of REACH Annex IX, a extended one-generation reproductive toxicity study does not need to be conducted since the repeated dose toxicity study does not indicate adverse effects on reproductive organs or tissues.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The substance CAS No. 1879-09-0 is one of the main components of the reaction mass of 2-tertbutyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol and it is present in a concentration of more than 70%. Therefore, the results obtained with the substance CAS No. 1879-09-0 can be used for the read-across approach.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

other: Read-across from an analogue

Sex:

male

Basis for effect level:

reproductive performance

Remarks on result:

other: read-across from an analogue for which NOEL = 150 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

other: read-across from an analogue

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: read-across from an analogue for which NOEL = 30 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

other: Read-across from an analogue

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other: read-across from an analogue for which NOEL = 30 mg/kg bw/day

Key result

Critical effects observed:

no

Reproductive effects observed:

not specified

Conclusions:

Based on read-across approach from experimental data on analogue substance 6-tert-butyl-2,4-xylenol (CAS 1879-09-0), the NOAELs of the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively.

Executive summary:

6-tert-Butyl-2,4-xylenol was studied in an OECD combined repeat dose and reproductive/developmental toxicity screening test (OECD Guideline 422) at doses of 0, 6, 30 and 150 mg/kg/day.The compound showed no effects on mating, fertility and oestrous cycle. In terms of reproductive/developmental toxicity, one female given 150 mg/kg died during the delivery. With three females in the same treatment group all pups died during the lactation period. A tendency to decrease of viability index of pups at Day 4 after birth was observed in 150 mg/kg group. The NOELs for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively. Based on these results, the NOAELs of the read-across approach was applied and the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

Males: 45 days including 14 days before mating
Females: from 14 days before mating to day 3 of lactation

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

(Vehicle)

Dose / conc.:

6 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Males,12; females, 12/group

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

Litter observations:

- No. of total live pups born
- Sex ratio
- No. of live pups on Day 4
- No. of dead pups born

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes
HISTOPATHOLOGY: Yes

Reproductive indices:

- Gestation index
- Implantation index
- Delivery index
- Live birth index

Offspring viability indices:

- Viability index on Day 4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical abnormal signs attributable to the administration of the test substance. However, two female animals given 150 mg/kg died at the end of gestation period (one of them during the delivery). In addition with three females of the same treatment group all pups died during the lactation period. A tendency for decrease in the viability index of the pups at Day 4 after birth was also observed in this group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Two female animals given 150 mg/kg died at the end of gestation period (one of them during the delivery)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain of females given 150 mg/kg was lower than that of the controls during the gestation period. However, body weights of males did not demonstrate any effects attributable to the administration of test substance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of both males and females did not demonstrate any effects attributable to the administration of test substance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination revealed swelling of centrilobular hepatocytes in males given 150 mg/kg and swelling and necrosis of centrilobular hepatocytes, and single cell necrosis in females given 150 mg/kg. The dead females and females with pups which all died showed increased incidences of parakeratosis of the tongue, esophageal swelling and necrosis of centrilobular hepatocytes, as well as a variety of degenerative charges, single cell necrosis and mitosis in the liver. Degeneration and protein cast in the proximal tubules and PAS positive granules deposited in the renal papilla, were observed in the kidneys of females given 150 mg/kg.

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The compound showed no effects on estrous cycle.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

The compound showed no effects on mating and fertility.

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A tendency for decrease in the viability index of the pups at Day 4 after birth was also observed in the group of females given 150 mg/kg bw.
Viability index on day 4 (%, mean ± SD) for males: from 95.5 ± 15.1 (non parametric analysis) (0 mg/kg) to 71.4 ± 42.9 (150 mg/kg)
Viability index on day 4 (% mean ± SD) for females: from 95.5 ± 15.1 (non parametric analysis) (0 mg/kg) to 69.7 ± 44.0 (150 mg/kg)
In 3 females of the same treatment group all pups died during the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A tendency for decrease in the viability index of the pups at Day 4 after birth was also observed in the group of females given 150 mg/kg bw.
Viability index on day 4 (%, mean ± SD) for males: from 95.5 ± 15.1 (non parametric analysis) (0 mg/kg) to 71.4 ± 42.9 (150 mg/kg)
Viability index on day 4 (% mean ± SD) for females: from 95.5 ± 15.1 (non parametric analysis) (0 mg/kg) to 69.7 ± 44.0 (150 mg/kg)
In 3 females of the same treatment group all pups died during the lactation period.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Critical effects observed:

no

Reproductive effects observed:

not specified

Conclusions:

NOELs for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively.

Executive summary:

6-tert-Butyl-2,4-xylenol was studied in an OECD combined repeat dose and reproductive/developmental toxicity screening test (OECD Guideline 422) at doses of 0, 6, 30 and 150 mg/kg/day.The compound showed no effects on mating, fertility and oestrous cycle. In terms of reproductive/developmental toxicity, one female given 150 mg/kg died during the delivery. With three females in the same treatment group all pups died during the lactation period. A tendency to decrease of viability index of pups at Day 4 after birth was observed in 150 mg/kg group. The NOELs for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively.

 

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with column 1 of REACH Annex IX, a extended one-generation reproductive toxicity study does not need to be conducted since the repeated dose toxicity study does not indicate adverse effects on reproductive organs or tissues.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1. This study was carried out in accordance with internationally valid GLP principles.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study: 6-tert-Butyl-2,4-xylenol was studied in an OECD combined repeat dose and reproductive/developmental toxicity screening test (OECD Guideline 422) at doses of 0, 6, 30 and 150 mg/kg/day.The compound showed no effects on mating, fertility and oestrous cycle. In terms of reproductive/developmental toxicity, one female given 150 mg/kg died during the delivery. With three females in the same treatment group all pups died during the lactation period. A tendency to decrease of viability index of pups at Day 4 after birth was observed in 150 mg/kg group. The NOELs for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively.Based on these results, the NOAELs of the read-across approach was applied and the reaction mass of 2-tert-butyl-4,6-dimethylphenol and 4-tert-butyl-2,5-dimethylphenol for reproductive performance of males and for that of females and pup development are considered to be 150 mg/kg/day and 30 mg/kg/day, respectively.

Data waiving: In accordance with column 1 of REACH Annex IX, a extended one-generation reproductive toxicity study does not need to be conducted since the repeated dose toxicity study does not indicate adverse effects on reproductive organs or tissues.

Justification for selection of Effect on fertility via oral route:

Only one study available.

Effects on developmental toxicity

Description of key information

Key study: OECD guideline 414 and EU method B.31. GLP study.

The NOAEL for both maternal toxicity and developmental toxicity was determined to be 50 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Medicine Centre at the Medical University in Białystok
- Age at study initiation: 13 weeks old
- Weight at study initiation: Average body weight of females of control group and treated groups: 232.5 g.
- Housing: Animals were kept in plastic cages with metal wire lid and dimensions: 37 x 22 x 15 cm (lenght x width x height) on UV sterilized bedding. At mating one female was placed in one cage with one male. At pregnancy the females were housed individually.
- Diet (e.g. ad libitum): “Murigran” standard laboratory fodder, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Minimum 3 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 30-80%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was diluted in corn oil directly before administration, in concentration correct for each dose.

A constant volume of solution (0.4mL of solution / 100 g bw) was used at every level of dosage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to confirm correct preparation of the test item, each of three doses was analyzed during the whole administration period using a liquid chromatographic method. The linearity of response of the analytical method, its specificity, precision, recovery, limit of quantification and detection were assessed in the process of the analytical method validation.

Details on mating procedure:

- If cohoused: The females were mated with not related males. The males came from the same husbandry and stock and were at a similar age.
- M/F ratio per cage: One male was used to mate with maximally three females.
- Length of cohabitation: from 1 to 4 days. In case of unsuccessful pairing the first male was replaced by a second one (4 cases), a third one (2 cases) or a fourth male (1 case).
- Proof of pregnancy: For each mating one female was placed in a cage with one male. Everyday in the morning, vaginal smears were taken from females. The sperm-positive females were accepted as mated and the day of observation of sperm in the smear was accepted as 0 day of gestation.

Duration of treatment / exposure:

The test item in treated groups and the corn oil in control group were given to pregnant females by gavage daily from 0 to 19th day of gestation.

Frequency of treatment:

Daily

Duration of test:

On day 20 of gestation (one day prior to the expected day of delivery), females were euthanised by intraperitoneal administration of morbital and subjected to caesarean section.

Dose / conc.:

8.3 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

No. of animals per sex per dose:

25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Sighting range-finding study:
Three different doses of the test item (10, 50 and 250 mg/kg bw) were administered orally to three groups of pregnant females. A concurrent control group of pregnant females was not given the test item. Seven pregnant females were used per group.

The doses were selected on the grounds of the LD50 value which is about 500 mg/kg bw (300 mg/kg bw: no mortality occurred; 2000 mg/kg bw: three animals died). The highest dose should be between 1/2-1/5 of the LD50, the medium dose should be between 1/10-1/20 of the LD50 and the lowest dose should be between 1/50-1/100 of the LD50.

The test item in the used doses administered from 5th to 19th gestation day, did not indicate any toxic influence on pregnant females, except for the decrease of food consumption from 5th to 8th in group 3. A statistically significant reduction on the weight of placenta was observed in the treated groups. No embryotoxicity, no fetotoxicity and no teratogenic effects were stated in the examined fetuses.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The evaluation of general condition of the animals, i.e. the observation of all animals for morbidity and mortality was performed twice a day during labour week and once a day on days off.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The detailed clinical observations were performed for all females on day of beginning of experiment (day 0) and then on 2nd, 5th, 8th, 11th, 14th, 17th and 20th day of gestation. The observations comprised: skin changes, coat changes, changes in eyes and mucous membranes, respiratory system, circulatory system, autonomous and central nervous system, somatic activity and behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the females were determined on days 0, 2, 5, 8, 11, 14, 17, and 20 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 0, 2, 5, 8, 11, 14, 17, and 20 of gestation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At necropsy, females were examined macroscopically for any abnormalities in body structure or pathological changes which could have influenced gestation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
After caesarean section, uterus from each female with fetuses was dissected. After removing of fetuses left and right uteri horn were examined. Number of viable and dead fetuses as well as number of resorptions was determined in each horn. Each pregnant uterus after removing of fetuses was weighed. Uteri of non pregnant females and non pregnant horns of uteri were fixed between two glass plates and overexposed by electric lamp to confirm the non-pregnant status. Then these uteri were stained using ammonium sulphide and then again overexpose with light by electric lamp in order to confirm the non-pregnant status. At the necropsy ovaries from each pregnant female were collected and fixed in 10% solution of formalin in order to determine number of corpora lutea.

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

Fetal examinations:

- External examinations: Yes. Sex of each fetus, body weight, length with tail and without tail was determined. Weight of fetuses with placenta and fetal membranes was determined first and then weight of fetuses without placenta and fetal membranes as well as weight of placenta were measured. After removal of fetal membranes each fetus was measured with tail and without tail, and detailed gross evaluation was performed.
During the gross evaluation attention was paid to:
- reactions to tactile impulses
- formation of body coverings
- formation of limbs
- number of fingers and toes
- shape of head
- formation of auricular conchas
- presence of nasal apertures
- formation of oral cavity
- presence of anus and tail
- Skeletal examinations: Yes. In every group, half of fetuses of each litter was subjected to evaluation of skeleton. The fetuses were fixed in 95% ethanol then overexposed in 1% KOH and stained with alizarin S, decolorized in Moll solution and moved to glycerine. The stained fetuses were examined and formation of skull bones, back-bone, ribs, acromial and pelvic girdles with limbs was evaluated. Points of ossification in sternum, metacarpus of fore limbs and metatarsus of hind limbs were counted during evaluation.
- Soft tissue examinations: Yes. The remaining fetuses from each litter were fixed in 10% solution of formalin, then cut in sagittal plane. Formation of particular cavities and presence of internal organs was evaluated.
- Head examinations: Yes.

Statistics:

Evaluation of the study was performed for pregnant females from which fetuses were obtained at necropsy. The obtained results (body weight of pregnant females, food consumption of pregnant females, number of corpora lutea, number of fetuses in litter, number of males and females in litter, weight of uteri, weight of fetuses, length of fetuses, number of ossification points in sternum and limbs) were elaborated statistically with the use of t-Student test with p ≤ 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Coat thinning in one female of group 1 and in one female of group 3 should not be connected with the test item. This sign occurred also in three females in control group and should be connected rather with hormonal changes during gestation. Clinical signs on the last day of experiment observed in one female of group 3 were connected with death of fetuses at the last period of gestation. No other clinical signs were stated in the females.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females survived during the period of experiment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant changes were observed in the mean body weights of the treated groups when compared to the control group at the end of the study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower food consumption in group 3 from 0 to 5th day of experiment was reflected in statistically significant lower body weight of females on 2nd and 5th day of gestation. Statistically significant greater food consumption from 8th to 11th day and from 14th to 20th day of experiment caused compensation of the low body weight of females at the initial period of experiment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

In group 3, in one female fifteen implantation sites and lack of fetuses were stated in uterus.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

In group 3, two resorptions in two females in group 3 were observed.

Early or late resorptions:

not specified

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

One dead fetus in group 1 should not be connected with test item and should be considered as incidental.
In group 3 in one female, in which on day of dissection some clinical signs were observed, only dead fetuses were stated.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Differences on average body weight of fetuses in treated groups compared to control group were observed but there is no relationship between the dose and the effect. Decreased body weight of fetuses without placenta was observed in group 1 and group 3. However, increased body weight of fetuses without placenta was observed in group 2. The lowest weight of fetuses without placenta was observed in group 3. Average weight of fetuses in control group, group 1 and group 2 are within the range from literature data, while average weight of fetuses without placenta in group 3 – 3.338 g is below the lower range (3.35 g to 3.85 g).

The length of fetuses with tail and without tail was lower in group 3. It may confirm delay of the growth of fetuses in group 3 considering the changes in body weight and length of fetuses.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

One dead fetus in group 1 should not be connected with test item and should be considered as incidental.
In group 3 in one female, in which on day of dissection some clinical signs were observed, only dead fetuses were stated.

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Dislocation of sternum ossification points and ribs attachment in one fetuses in group 1 should not be connected with test item. This case was noticed only in one fetus and similar changes in ribs and sternum were observed in fetuses of control groups in other prenatal developmental toxicity studies performed at the testing facility. Therefore, this change should be considered as a spontaneous disorder.

Decrease in number of ossification points is considered as a change associated with delayed development due to toxicity which does not influence postnatal development and is later compensated. A statistically significant lower number of ossification points of metacarpus in group 1 results from greater percentage of fetuses with 3 ossification points of metacarpus in group 1, whereas lower number of ossification points of metatarsus in group 3 results from the fact that in one female of this group 4 fetuses had 3 ossification points of metatarsus and 1 fetus had no ossification points of metatarsus. All other fetuses in group 3 had 4 ossification points of metatarsus. Additionally, in control group, which treated groups are compared to, average number of ossification points of metatarsus was above 4, because 7 fetuses from one female in control group had 5 ossification points of metacarpus (likewise in group 2).

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

In each of the treated groups one case of conjoined placenta was stated. This finding is not considered to be treatment related. Subcutaneous blood hemorrhages observed in fetuses in different parts of the body occurred in control group and in treated groups and should not be connected with the test item. Subcutaneous blood hemorrhages were observed often in fetuses in other prenatal developmental toxicity studies performed in the testing facility and they should be considered as typical disturbances in fetuses.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Developmental effects observed:

not specified

Table 1 - Results of mating - main study

 

Parameter	Number of females
	Group 0	Group 1	Group 2	Group 3
Number of females per group	25	25	25	25
Number of pregnant females	21	23	22	20
Number of not pregnant females	4	2	3	5
Females with all dead fetuses	-	-	-	1
Females with implantations and without fetuses	-	-	-	1
Females with resorptions	6 [1,10,17,18, 19(2),20]	3 [2,11,24]	4 [3,7,24(4),25]	2 [8,21]
Number of females for evaluation	21	23	22	18

[ ] computer numbers of females

( ) number of resorptions

Table 2 - Body weights of females (g) - main study

Day of gestation	Group
	0	1	2	3
0	233.71 ± 20.83	235.13 ± 20.47	230.23 ± 15.38	230.61 ± 15.44
2	237.48 ± 20.52	237.30 ± 23.32	232.05 ± 16.27	219.83 ± 14.26*
5	244.67 ± 20.29	248.13 ± 21.41	245.77 ± 15.78	228.67 ± 21.77*
8	253.14 ± 21.77	254.00 ± 21.68	252.18 ± 18.22	243.06 ± 20.82
11	264.90 ± 19.68	267.13 ± 23.76	265.95 ± 17.66	258.44 ± 25.22
14	275.29 ± 20.12	277.04 ± 22.44	278.45 ± 17.22	274.94 ± 20.76
17	300.57 ± 22.66	301.57 ± 23.53	304.14 ± 20.99	300.67 ± 23.53
20	335.81 ± 24.24	340.70 ± 25.87	340.64 ± 25.65	339.78 ± 30.57

* statistically significant difference with p ≤ 0.05

Table 3 - Food consumption (g/100 g bw/day) - main study

Day of gestation	Group
	0	1	2	3
0 – 2	5.7 ± 1.84	5.1 ± 1.38	4.8 ± 1.08	2.2 ± 1.13 *
2 – 5	7.2 ± 1.29	7.0 ± 0.86	7.2 ± 0.93	4.7 ± 1.91 *
5 – 8	7.1 ± 0.98	7.0 ± 1.01	7.2 ± 0.84	7.3 ± 1.26
8 – 11	6.8 ± 0.85	6.9 ± 1.02	6.9 ± 0.79	7.8 ± 1.12 *
11 – 14	6.9 ± 0.93	6.7 ± 0.88	6.7 ± 0.81	7.4 ± 1.52
14 – 17	6.7 ± 1.06	6.6 ± 0.79	6.8 ± 0.84	7.6 ± 0.67 *
17 – 20	6.4 ± 0.64	6.6 ± 0.68	6.6 ± 0.59	6.9 ± 0.85 *

  * statistically significant difference with p ≤ 0.05

Table 4 - Average weight of uterus of pregnant females - main study

Group	Number of pregnant females	Weight of uterus
0	21	5.19 ± 0.84
1	23	5.51 ± 0.75
2	22	5.12 ± 1.14
3	18	5.08 ± 1.31

Table 5 - Number of fetuses - main study

Group	Number of pregnant females	Number of fetuses
		Total	in litters min. – max.	average in litter in 1 female	females	males	dead
0	21	247	2 – 16	11.76 ± 2.91	5.76 ± 2.28	6.00 ± 2.17	0
1	23	270	6 – 15	11.74 ± 1.94	6.22 ± 2.00	5.52 ± 1.86	1
2	22	244	5 – 14	11.09 ± 2.39	6.45 ± 2.09	4.64 ± 1.94	0
3	18	199	1 – 15	11.06 ± 3.69	5.39 ± 1.91	6.38 ± 1.41	13*

*- all fetuses dead from one female (female not accounted in statistical evaluation)

Table 6 - Number of implantations and number or resorptions - main study

Group	Number of pregnant females	Average number of corpora lutea per one female	Number of implantations	Number of resorptions
				total	in females min - max	average in one female
0	21	13.00 ± 1.61	0	7	0 – 2	0.33 ± 0.58
1	23	12.57 ± 1.62	0	3	0 – 1	0.13 ± 0.34
2	22	12.55 ± 1.63	0	7	0 – 4	0.32 ± 0.89
3	18	13.11 ± 1.91	15*	2	0 – 1	0.11 ± 0.32

*- all implantations from one female (female not accounted in statistical evaluation)

 

Table 7 - Average weight of fetuses and placenta (g) - main study

Group	Number of examined fetuses	Weight of fetuses		Weight of placenta
		with placenta (x)	without placenta
0	247	4.706 ± 0.607	3.431 ± 0.535	0.535 ± 0.098
1	270	4.627 ± 0.364	3.354 ± 0.307 *	0.535 ± 0.086
2	244	4.884 ± 0.437 *	3.467 ± 0.442 *	0.555 ± 0.115 *
3	199	4.809 ± 0.604	3.338 ± 0.512 *	0.557 ± 0.180

*statistically significant difference with p ≤ 0.05

(x) with placenta and fetal membranes

Table 8 - Average length of fetuses and placenta (cm) - main study

Group	Number of examined fetuses	Length of fetuses
		with tail	without tail
0	247	5,32 ± 0,24	3,75 ± 0,20
1	270	5,28 ± 0,23	3,74 ± 0,17
2	244	5,30 ± 0,27	3,78 ± 0,21
3	199	5,18 ± 0,34 *	3,69 ± 0,26 *

 * statistically significant difference with p ≤ 0.05

Table 9 - Pathological changes in fetuses - main study

Pathological changes	Number of fetuses
	Group 0	Group 1	Group 2	Group 3
Total number of fetuses	247	271	244	212
Number of alive fetuses	247	270	244	199
Number of dead fetuses	0	1[7]	0	13*[3]
Number of blood hemorrhage	6	6	5	3
Blood hemorrhage on back	1  [5]	3 [4, 6, 17]	5  [3, 6(2), 10(2)]	3  [6 (3)]
Blood hemorrhage on metatarsus	1  [4]	-	-	-
Blood hemorrhage on thigh	-	3  [2(2), 6]	-	-
Blood hemorrhage on shank	1  [8]	-	-	-
Blood hemorrhage on arm	1  [6]	-	-	-
Blood hemorrhage on forearm	1  [6]	-	-	-
Blood hemorrhage on tail	1  [4]	-	-	-
Conjoined placenta	-	1  [7] ♀** i ♂	1  [10] ♂ i ♂	1  [22] ♀ i ♂

 *–all fetuses dead from one female (female not accounted in statistical evaluation)

**– dead fetuses

[ ] –computer numbers of females

( )–number of fetuses in a given female, if more than 1

Table 10 - Pathological changes in skeletal system - main study

 

Pathological changes	Number of fetuses
	Group 0	Group 1	Group 2	Group 3
No ossification points of sternum	-	-	-	1  [10]
1 ossification point of sternum	-	-	-	2  [10(2)]
2 ossification points of sternum	-	-	-	2  [10(2)]
3 ossification points of sternum	5 [10(3),14,16]	1 [21]	2 [16(2)]	3 [8(2),12]
Dislocation of sternum ossification points and rib attachment	-	1  [10]	-	-
No ossification points of metatarsus	-	-	-	1  [10]
3 ossification points of metatarsus	-	-	-	4  [10]
1 ossification point of metacarpus	-	-	-	1  [10]
5 ossification points of metatarsus	7 [25(7)]	-	7 [17(7)]	-
Total number of all pathological changes (Table 22 and 23)	18	9	15	18

[ ] computer numbers of females

( ) number of fetuses in a given female, if more than 1

Table 11 - Percentage of fetuses with a given number of ossification points of sternum (%) - main study

Group	Number of examined fetuses	Number of ossification points
		0	1	2	3	4	5	6	7
0	129	0.0	0.0	0.0	3.88	14.73	40.31	41.08	0,0
1	141	0.0	0.0	0.0	0.71	14.89	54.61	29.79	0,0
2	129	0.0	0.0	0.0	1.55	13.95	41.86	42.64	0,0
3	105	0.95	1.90	1.90	2.86	12.38	35.24	44.77	0,0

Table 12 - Percentage of fetuses with a given number of ossification points of limbs (%) - main study

Group	Number of examined fetuses	Fore limbs Number of ossification points						Hind limbs Number of ossification points
		0	1	2	3	4	5	0	1	2	3	4	5	6	7
0	129	0.0	0.0	0.0	39.53	60.47	0.0	0.0	0.0	0.0	0.0	94.57	5.43	0.0	0.0
1	141	0.0	0.0	0.0	58.87	41.13	0.0	0.0	0.0	0.0	0.0	100,0	0.0	0.0	0.0
2	129	0.0	0.0	0.0	47.29	52.71	0.0	0.0	0.0	0.0	0.0	94.57	5.43	0.0	0.0
3	105	0.0	0.95	0.0	39.05	60.00	0.0	0.95	0.0	0.0	3.81	95.24	0.0	0.0	0.0

Table 13 - Average number of ossification points of sternum and limbs - main study

Group	Number of examined fetuses	Sternum	Metacarpus of fore limbs	Metatarsus of hind limbs
0	129	5.19 ± 0.83	3.60 ± 0.49	4.05 ± 0.23
1	141	5.13 ± 0.68	3.41 ± 0.49 *	4,00 ± 0,00
2	129	5.26 ± 0.75	3.53 ± 0.50	4.05 ± 0.23
3	105	5.09 ± 1.19	3.58 ± 0.55	3.92 ± 0.43 *

 * statistically significant difference with p ≤ 0.05

Conclusions:

The NOAEL for both maternal toxicity and developmental toxicity was determined to be 50 mg/kg bw/day.

Executive summary:

A prenatal developmental toxicity study on rats was performed according to OECD guideline 414 and EU method B.31 in order to determine test item influence and possible toxic effects to pregnant females and their fetuses due to treatment during period of gestation. The study was conducted on 100 females divided into four experimental groups: three treated groups and one control group. Every group was consisted of 25 females. From day 0 to 19th of gestation the females of treated group were given by gavage the test item dissolved in corn oil. The following doses of the test item were used: in group 1 – 8.3 mg/kg bw/day, in group 2 – 50 mg/kg bw/day and in group 3 – 300 mg/kg bw/day. Females of the control group (group 0) were given corn oil. During the study, clinical observations for mortality and signs of toxic influence of the test item were performed in females. Body weight and food consumption were recorded. On Day 20 of gestation, the females were killed and caesarean section with gross examination was performed in each female. The following endpoints were determined in each pregnant females in the sighting study and the main study: number of alive and dead fetuses, number of resorptions. After the removing of fetuses each uterus was weighed. The non-gravid uteri and non-gravid horns were overexposed by electric lamp and were stained using ammonium sulphide in order to confirm the non-pregnant status. In the main study the number of corpora lutea was determined in fixed ovaries. The following endpoints were determined in all fetuses: sex, body weight with and without placenta, weight of placenta, body length with and without tail. Each fetus was subjected to gross examination. Half of fetuses from each litter was stained with alizarin and subjected to evaluation of skeleton. The rest of them was fixed in 10% solution of formalin and evaluated for formation of body cavities and internal organs. Based on the results, the dose levels 8.3 and 50 mg/kg bw /day did not indicate any toxic influence on pregnant females. The dose level of 300 mg/kg bw/day caused increased intrauterine mortality and reduction of body weight and body length in fetuses. The dose levels 8.3, 50 and 300 mg/kg bw/day did not cause morphological changes in fetuses of treated females.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1. This study was carried out in accordance with internationally valid GLP principles.

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

Key study: OECD guideline 414 and EU method B.31. GLP study.

The NOAEL for both maternal toxicity and developmental toxicity was determined to be 50 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:

Only one study available

Justification for classification or non-classification

Based on the available data, the substance is not classified as toxic for reproduction.