Bumetrizole

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

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:

guideline study

Reason / purpose for cross-reference:

reference to same study

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): bumetrizole and 2-(2'-Hydroxy-3-tert -butyl-5’-methyphenyl) -5-chlorobenzotriazole
- Appearance/physical state: light yellow/powder
- Analytical purity: 99.9% w/w
- Lot/batch No.: 01721IW4
- Stability under test conditions: Stable
- Storage condition of test material: Stored sealed in a cabinet at 20.0 - 25.3° C
- Supplier: Ciba Specialty Chemicals, Osaka, Japan

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river Laboratories Japan (Hino Breeding Center)
- Age at study initiation/start of treatment: 7 weeks/10 weeks
- Weight at study initiation: Test group males 230 - 249 g; Test group females 176 - 202 g; Recovery group females 179 - 198 g
- Fasting period before study: Day before administration
- Housing: Individually
- Diet: CRF-1 (Oriental Yeast Co.), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days quarantine, 16 days acclimation (17 days for recovery group)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 40 - 56 (on 5 occasions, deviations below 40% humdity were recorded. As humidity deviation was slight (36 - 39.8%) and humidity for other period was within allowable limits, it is considered that this deviations had no influence to the test results.
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From May 13th, 2005 to July 13-15th (main group) and July 27-28th, 2005 (recovery group)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % w/v methyl cellulose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
After the necessary quantity of bumetrizole was weighed (Electronic balance: AT250 and PM2500, METTLER-TOLEDO), the specified concentration was prepared by suspension in 0.5% (w/v) methylcellulose solution using a conditioning mixer (AR-250, THINKY). Besides, for preparation, the test material was not adjusted according to the content, and administration dose was indicated as bulk powder weight.

VEHICLE
- Lot/batch no: Lot No. 110654
- Concentration in vehicle: 1.25, 5 and 20%
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): After day 18 of pregnancy, females were housed individually in a plastic cage until day 4 of lactation, then in a stainless steel cage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test material in the dosing material used for administration was determined by HPLC on the first and last days of administration.

Duration of treatment / exposure:

Males: Total of 42 days (from 14 days before mating to 28 days after mating)
Females: Total of 44 - 56 days (from 14 days before mating, during pregnancy and up to lactation day 6)

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Test group (male): 12/dose (6 were assigned to the recovery group)
Test group (female): 12/dose
Recovery group females: 6/group at control, 250 or 1000 mg/kg bw/day.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses selected on the basis of a preliminary repeated dose study (no adverse influence on mortality, clinical signs, food consumption or necropsy findings at 1000 mg/kg bw/day).
- Rationale for animal assignment: random
- Post-exposure recovery period: Males - half the animals in each dose group were allowed a recovery period of 14 days after the administration period; Recovery group females - 14 day recovery period after a 42 day administration period.

Parental animals: Observations and examinations:

Refer to Section 7.5.1 Repeated dose toxicity: oral for details

Oestrous cyclicity (parental animals):

The estrus cycle of the test group females was observed once a day from the start of the administration period until the day before copulation verification. When the estrous cycle was observed for two consecutive days, it was counted as one.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (birthday and on day 4 after birth)

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

Refer to Section 7.5.1 Repeated dose toxicity: oral for details

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

The test of significance was performed between control groups and each test group and recorded separately at p<0.05 and p<0.01. Unimpregnated females after copulation were excluded from the counting of general signs, body weight, food consumption and FOB. For body weights of pups, the average of one litter defined as one unit, the average and total value of one litter were calculated.

Averages and standard deviations in each group were calculated for body weights (parent animals, pups), food consumption, frequency of rearing and grooming for Functional Observational Battery (FOB), grip strength, spontaneous motor activity, urinary volume, urinary specific gravity, records of hematological/blood chemical examination, absolute and relative weight of organs, estrus frequency, copulation required days, pregnancy period, number of corpora lutea, number of implantations, implantation index, total number of pups born, number of live pups, number of stillborns, delivery index, rate of pups delivery, live birth index, live pups on lactation day 4, viability index, occurrence rate of abnormal appearance. Subsequently, the test of distribution uniformity by Bartlett's test performed, and the Dunett Test was done when the distribution is uniform. In contrast, the Dunett Test using rank was done when the homoscedastic was not recognized.

For the Functional Observational Battery (FOB)(however, excluded frequency of rearing and grooming) and sensory response, the average and range in each group were calculated. Then, the Dunett Test using rank was done.

The copulation index, fertility index, and gestation index were calculated by chi-square test.

Reproductive indices:

Copulation required days
Copulation index [(number of pairs with successful copulation/number of pairs mated) x 100]
Fertility index [(number of pregnant females/number of pairs with successful copulation) x 100]
Gestation index [(number of females with live pups/number of pregnant females) x 100]
Pregnancy period [delivery date (lactation day 0 – copulation verification day)]
Number of corpora lutea
Number of implantations
Implantation index [(number of implantations/number of corpora lutea) x 100]

Offspring viability indices:

Total number of pups born (number of live pups + number of stillborns)
Number of live pups
Number of stillborns
Delivery index [(number of pups born /number of implantations) x 100]
Rate of pups delivery [(number of live pups on day 0/number of implantations) x 100]
Live birth index [(number of live pups on day 0/ number of pups born) x 100]
Survival pups on lactation day 4
Viability index [(number of live pups on lactation day 4/number of live pups on lactation day 0) × 100]
Occurrence rate of abnormal appearance [(number of pups with abnormal appearance/number of neonates) x 100]

Refer to Section 7.5.1 Repeated dose toxicity: oral for details of general toxicity effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant difference was observed in the estrus frequency during the administration period (14 days) before the start of mating in administered groups compared to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Copulation required days, copulation index, number of pregnant females, fertility index: All animals copulated in every group. The copulation index was 100 %. No significant difference was observed in the copulation required days between administered groups and controls. There was one case of an unimpregnated female in both the controls and the 100 mg/kg bw/day group. No significant difference was observed in fertility index between the administered groups and the controls.

Pregnancy period: No significant differences between administered groups and controls.

Number of corpora lutea, number of implantations and implantation index: No significant difference was observed in the number of corpora lutea, number of implantations and implantation index in 1000 and 250 mg/kg bw/day groups compared to the control group. Significant high value of implantation index was seen in 62.5 mg/kg bw/day group compared to the control group, however because this effect was not dose related, it was considered to not be due to administration of the test material.

Gestation index, delivery conditions, nursing conditions: Gestation index was 100 % in all groups. No abnormality was observed in the delivery conditions and in the lactation conditions in any group.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Highest dose tested. No effects seen in the reprodcutive parameters up to the highest dose tested.

Critical effects observed:

no

VIABILITY (OFFSPRING)
There was no siginificant difference in viability index between any dose group and the controls.

CLINICAL SIGNS (OFFSPRING)
There was no significant difference in clinical signs between any dose group and controls. There was one acaudal pup in the 1000 mg/kg bw/day group, but this was considered to be a natural occurrence and not related to administration of the test material.

BODY WEIGHT (OFFSPRING)
No significant difference was observed in dose groups compared to controls in the average body weight by sex on lactation day 0 or 4, the average body weights per litter on lactation day 0 or 4, and the total body weights per litter on lactation day 0 or 4.

SEXUAL MATURATION (OFFSPRING)
No data

ORGAN WEIGHTS (OFFSPRING)
No data

GROSS PATHOLOGY (OFFSPRING)
In pups born dead, no significant difference was observed in the 1000 and 62.6 mg/kg bw/day groups as well as in the controls. Though an absence of intraabdominal organ was observed in one case in the 250 mg/kg bw/day group, as it was not relevant to the administration dose, it was not considered to be due to administration of the test material.

In live pups examined at the end of the study, no abnormalities were observed in any group.

HISTOPATHOLOGY (OFFSPRING)
No data

OTHER FINDINGS (OFFSPRING)
Total number of pups born, number of dead pups, number of live pups on day 0, sex ratio on day 0, delivery index, rate of pups delivery, live birth index: No significant difference between controls and 1000 or 62.5 mg/kg bw/day dose groups in any of these parameters. Delivery index and rate of pups delivery were significantly lower than controls at 250 mg/kg bw/day, however as the effect was not dose related, it was considered to not be due to administration of the test material.

Sex ratio on day 4: No significant difference between controls and any dose group.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested. No effects seen in developmental parameters examined at the highest dose tested

Critical effects observed:

no

Reproductive effects observed:

no

Please refer to Section 7.5.1 for tables related to repeat dose toxicity in parental animals

2)  Reproductive Developmental Toxicity

Administration dose	Control group	Low Dose Group	Medium Dose group	High Dose Group
(mg/kg)	0	62.5	250	1000
Parental animal
Estrus frequency	3.1	3.5	3.4	3.3
Copulation rate (%)	100.0	100.0	100.0	100.0
Conception rate (%)	91.7	100.0	100.0	91.7
Number of corpus luteum of    pregnancy	16.4	15.6	17.3	16.5
Number of implantations	14.4	15.0	15.7	15.4
Implantation rate (%)	88.0	96.3↑↑^a)	90.3	93.7
Number of pregnant dams	11	12	12	11
Number of dams with live births	11	12	12	11
Birth rate (%)	100.0	100.0	100.0	100.0
Pregnancy period	22.3	22.5	22.5	22.3
Neonate
Number of total births	13.8	13.8	14.0	14.7
Delivery rate (%)	95.2	89.7	85.5↓↓^a)	93.5
Rate of pups delivery (%)	95.3	89.7	85.5↓↓^a)	93.5
Number of survival pups   (lactation day 0 )	13.6	13.4	13.4	14.4
Survival birth rate (%)	98.9	97.0	95.8	97.5
Sex ratio (♂♀, lactation day 0)	1.06	1.34	2.24	1.21
Number of survival pups  (lactation day 4)	13.5	13.1	13.3	14.2
Survival rate (%)	98.6	97.5	98.9	98.7
Sex ratio(♂♀, lactation day 4)	1.06	1.43	2.25	1.21
Body weights (lactation day 0)		-/-	-/-	-/-
Body weights (lactation day 4)		-/-	-/-	-/-
Abnormality of appearance (N)	-	-	-	Acaudal (1)^b)
Necropsy findings (N)	-	-	Interperitoneal fracture (1)^b)	-
NOAEL	Parental animal: = 1000 mg/kg/day for both male and female rats. Pups: = 1000 mg/kg/day.
Change for estimated grounds of NOAEL	Because no effect was observed in any parameter at 1000 mg/kg administration for male rats. Because no effect was observed in any parameter at 1000 mg/kg administration for female rats. Because no effect was observed in any parameter at 1000 mg/kg administration for pups.

NOAEL: No Observed Adverse Effect Level

-/- :♂/♀

Copulation rate (%) = (Number of copulation verified animals/Number of hybridization animals) x100

Conception rate (%) = (Number of pregnant animals/Number of copulation verified animals) x100

Implantation Rate (%) = (Number of implantations/Number of corpus luteum of pregnancy) x100

Birth rate (%) = (Number of dams with live births/Number of pregnant dams) x100

Pregnancy period = Nursing day 0 [Delivery verified day] (date) - Pregnancy day 0 (date)

Delivery rate (%) = (Number of total births/Number of implantations) x100

Pups birth rate (%) = (Number of neonatal pups on lactation 0 day/Number of implantations) x100

Survival birth rate (%) = (Number of survivals on lactation day 0/ Number of total births) x100

Survival rate (%) = (Number of survivals on lactation day 4/Number of survivals on lactation day 0) x 100

-: No change, or no significant difference with control group.

↑↑ ↓↓: 5% significant difference

a): Judged as Not Toxicological Effect.

b): Judged as contingent case.

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Incomplete characterization of the test substance e.g. no data on test material purity.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG 478 Genetic Toxicology: Rodent Dominant Lethal Test

Deviations:

yes

Remarks:

(only two dose levels were applied)

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): TK10048
- Lot/batch No.: EN 26580(1/75)

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1.0
- Humidity (%): 50±5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on mating procedure:

Each group consisted of 20 males, each of which was placed in a cage with 2 untreated females immediately after treatment. At the end of 1 week, the females were removed from the cages and replaced by another group of 2 females. The procedure was continued for 6 consecutive weeks. The females were daily examined for successful mating indicated by the occurrence of a vaginal plug. The day that the vaginal plug was observed was designated as "Day 0" of gestation.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

single dose application

Frequency of treatment:

once

Dose / conc.:

1 000 other: mg/kg bw (actual dose received)

Dose / conc.:

3 000 other: mg/kg bw (actual dose received)

No. of animals per sex per dose:

20 males
40 females

Control animals:

yes, concurrent vehicle

Positive control:

No positive controls were used in this particular study. However, the same laboratory that conducted this study also conducted studies on positive control substances at the same facility using the same strain of mice. The results of these tests have been published (see, for example, Fritz H et al (1973) Agents and Actions, 3, 35). This study clearly demonstrates that dominate lethal effects can be chemically induced in NMRI mice.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 000 mg/kg bw (total dose)

Basis for effect level:

other: No significant differences in mating ratio, numbers of implantations or embryonic deaths between the mating groups.

Critical effects observed:

no

Reproductive effects observed:

no

No evidence of dominant lethal effect was observed in the progeny of male mice treated with TK 10048 Table 1: Dominant lethal assay results Dose Group (mg/kg) No. females mated* No. of pregnant females No. of implantations Live embryos Embryonic deaths Total % Total % Total Mean SD Total % Total % Mating Period I 0 38 95.0 36 94.7 408 11.33 3.80 365 89.5 43 10.5 1000 39 97.5 34 87.2 391 11.50 4.32 370 94.6 21 5.4 3000 33 82.5 21 63.6 230 10.95 3.90 213 92.6 17 7.4 Mating Period II 0 37 92.5 34 91.9 399 11.74 4.19 373 93.5 26 6.5 1000 40 100 36 90.0 481 13.36 2.32 455 94.6 26 5.4 3000 38 95.0 30 78.9 383 12.77 3.18 352 91.9 31 8.1 Mating Period III 0 40 100 34 85.0 413 12.15 3.11 388 94.0 25 6.0 1000 39 97.5 33 84.6 434 13.15 1.73 414 95.4 20 4.6 3000 40 100 32 80.0 373 11.66 2.62 358 96.0 15 4.0 Mating Period IV 0 38 95.0 30 78.9 388 12.93 1.53 357 92.0 31 8.0 1000 37 94.9 34 91.9 455 13.38 1.39 433 95.2 22 4.8 3000 35 87.5 30 85.7 384 12.80 1.67 354 92.2 30 7.8 Mating Period V 0 36 90.0 33 91.7 390 11.82 2.56 371 95.1 19 4.9 1000 38 95.0 34 89.5 442 13.00 1.74 415 93.9 27 6.1 3000 34 85.0 31 91.2 388 12.52 1.52 358 92.3 30 7.7 Mating Period VI 0 36 90.0 29 80.6 364 12.55 2.89 344 94.5 20 5.5 1000 36 90.0 32 88.9 400 12.50 1.97 371 92.8 29 7.2 3000 39 97.5 31 79.5 363 11.71 2.75 333 91.7 30 8.3 Cumulative control 328 - 281 85.7 3242 11.54 2.58 2998 92.5 244 7.5 * 40 females/group

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:

REACH allows the assessment of the reproductive toxicity of a given chemical with the help of findings from studies with repeated administration. This is in line with the idea that the information requirements under REACH are regarded as the evaluation of endpoints which does not necessarily require data from specific studies. The test substance was tested in a OECD 422 study. This study did not show any concern regarding reproductive toxicity of the test substance. Thus, a two-generation study is regarded as redundant. This waiving argument is in line with the guidance document R7a and is further justified as followed: Because of a high correlation, histopathology data and organ weights from repeated dose studies may be used to assess male fertility (Mangelsdorf, 2003). These parameters, taken from 90 day studies, were in fact shown to be more sensitive than fertility parameters measured during multi-generation studies. It could also be shown that exposure for 4 weeks suffices for an assessment of male fertility, although 90 day studies have been regarded as superior in the past because they cover a complete cycle of spermatogenesis (Mangelsdorf, 2003). If such a 28 day study shows neither relevantly elevated testis or ovary weights nor histopathological alterations in those organs, the weight of the evidence is that effects on reproduction are also not expected (BAuA Forschungsbericht Fb 984, 2003). A comparison of more than one hundred 90 day studies with two-generation studies that used the same test substance additionally showed that the NOAELs differed by less than the variation limit of studies, i.e. a factor of two (Janer, 2007). Therefore, the information gained from a two-generation study can be regarded as minimal if a 90 day study has been performed. Accordingly, there is no need for evaluation on a second species. References: - BAuA (2003). Extrapolation from results of animal studies to humans for the endpoint male fertility. Forschungsbericht Fb 984. - Janer G, Hakkert BC, Piersma, AH, Vermeire T, Slob W (2007). A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study. Reproductive Toxicol 24: 103-113 - Mangelsdorf I, Buschmann J, Orthen B (2003). Some aspects relating to the evaluation of the effects of chemicals on male fertility. Reg Toxicol Pharmacol 37: 356-369

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD Test Guideline 422), Crj: CD (SD) rats (both sexes) were treated with the test substance (99.9% pure) at dose levels of 62.5, 250 or 1000 mg/kg bw/day by gavage. Control animals received the vehicle (0.5% w/w methylcellulose). The males (12 per dose) were dosed beginning 14 days before mating and continuing until 28 days after mating (a total of 42 days). The females (12 per dose) were dosed beginning 14 days before mating, during pregnancy and up to lactation day 6 (a total of 44 -56 days). After treatment, 6 of the12 treated males were allowed a recovery period of 14 days. For the females, a satellite group of 6 animals was maintained for additional 14 days after treatment for controls and the mid and high dose levels.

There were no test substance related effects on food consumption, functional observational battery (FOB), sensory response examination, grip strength, spontaneous motor activity measurements, clinical chemistry, macroscopic and microscopic examinations including organ weight.

In the parental animals, there were no statistically different or toxicologically relevant changes in the reproductive performance. Number of days to copulation, copulation index, number of pregnant females, fertility index, gestation duration, oestrous cycle, number of corpora lutea, number of implantations and implantation index as well as the delivery and nursing conditions were comparable between all treated groups and the controls. The NOAEL for fertility is 1000 mg/kg bw/day.

In the offsprings, there were also no toxicologically relevant changes in pup viability and development noted. Mortality, clinical signs, body weight changes and gross pathology findings were comparable between all treated groups and the controls. On the basis of the observed parameters, a NOAEL for developmental toxicity of 1000 mg/kg bw/day was estimated (Nihon Bioresearch Center Inc., 2007).

This study is acceptable as it satisfies the guideline requirement for a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD Guideline 422) and was performed under GLP.

 

In line with the results of the above OECD 422 study are the results of a dominant lethal assay in which the reproduction performance and fertility of male animals treated with the test substance (single dose application of 1000 and 3000 mg/kg bw) was not impeded (Ciba Geigy Ltd. 1977). For further information see the chapter on gene mutation.

Short description of key information:
No indication of fertility impairment was seen in rats (male and female) after treatment for 42 - 56 days with the test substance by gavage. A NOAEL for fertility of 1000 mg/kg bw was derived.

Effects on developmental toxicity

Description of key information

Developmental and teratogenic effects were not detected in the rat and at doses that were not significantly toxic to the parent animals.
- NOAEL (parental) = 3000 mg/kg bw/day
- NOAEL (developmental) = 3000 mg/kg bw

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Treatment of dams was limited to gestation periods Day 6-15 of gestation only as opposed to a day before scheduled kill - certain maternal parameters not checked e.g corpora lutea was not determined.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(test substance administered on day 6-15 of gestation only, certain maternal parameters not checked e.g corpora lutea was not determined)

Principles of method if other than guideline:

The test compound was administered orally to pregnant rats from day 6 until 15 of gestation. Dams were observed during the pregnancy and then killed on day 21 of the pregnancy and an autopsy performed to examine the dam and the foetuses.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): TK 10048
- Physical state: solid
- Lot/batch No.: EN 26580 (1/75)

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Closed SPF breeding colony
- Age at study initiation: not stated
- Weight at study initiation: 240 g
- Fasting period before study: not stated
- Housing: Macrolon cages
- Diet: ad libitum; Nafag No. 890
- Water: ad libitum; tap water
- Acclimation period: not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 0.5
- Humidity (%): 60 ± 5
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

(2%, aqueous)

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Amount of vehicle (if gavage): 1mL/100 g bw
- Lot/batch no. (if required): not stated
- Purity: not stated

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:3
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes, the albino rats were Sprague -Dawley derived and obtained from a closed SPF breeding colony
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 until day 15 of pregnancy inclusive.

Frequency of treatment:

once daily

Duration of test:

21 days

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not stated
- Rationale for animal assignment (if not random): not stated

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily

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, a mean food consumption graph was present in the study, but no details about individual animal food consumption.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No, as not a feeding study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Assessment of the dam's organs, especially the ovaries and uterus.

OTHER:The foetuses were subjected to careful external inspection and the condition of their body orifices was checked. They were then weighed individually followed by examination of the viscera and skeletal assessment.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Not stated
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
-Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: No

Statistics:

Some statistics were present in the tables, although not in much detail. Standard deviation was only applied to weight of live foetuses. Confidence limits of the CMC-control for the skeletal assessment was present.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No reactions to treatment were observed. Body-weight gain and food consumption were comparable for all groups.

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The implantation ratio and embryolethality were comparable for all groups. The average body weights of foetuses in each group were not significantly different from controls. Histopathology revealed the following minor anomalies: Two foetuses of one litter from the 300 mg/kg dose group showed either hypoplasia of the lungs or slight oedema-like changes of subcutaneous tissue (anasarca). One foetus from the 3000 mg/kg dose group and one from the vehicle control also showed hypoplasia. The aforementioned types of minor anomalies were also found in the cumulative control at incidences of 0.4% (hypoplasia of lungs) and 1.3% (anasarca). Based on these findings, no substance-related effect on embryonic or foetal development is assumed.

Skeletal assessment did not reveal any clearcut differences between the foetuses of the experimental group and those of the vehicle control.

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Results of uterus examinations. 

	Dose groups (mg/kg bw)
	Historical controls	300	1,000	3,000	Control
No. of dams	510	25	25	25	25
Spontaneous deaths	0	0	0	0	0
No of females with implantations	453^a	24	24	24	23
Mean No. of implantations	13.18	14.33	14.13	13.88	12.87
Embryonic resorptions (%)	6.7	9.0	11.2	7.2	11.8
Foetal resorptions (%)	0.1	0.3	0.3	0	0.3
Dead foetuses (%)	0.1	0.6	0	0	0.3
Live foetuses (%)	93.1	90.1	88.5	92.8	87.9
Live foetuses with malformations	10 / 5,546^b	0 / 310	0 / 300	0 / 309	0 / 259
Mean weight of live foetuses (g)	5.25 ± 0.44	5.19 ± 0.39	5.18 ±0.38	5.28 ± 0.39	5.20 ± 0.45

 

(a) Including one female with totally aborted litter (not taken into further consideration of data)

(b) Two cleft palates (one associated with mandibular hypoplasia), 2 mandibular aplasias, 3 general oedemas, 3 “open eyes”

Table 2: Skeletal changes in the foetuses.

Dose group (mg/kg bw)	No. of skeletons examined	Phalangeal nuclei^a		Calcaneus^a	Sternebrae^d	Vertebrae^e	Vertebrae^f	Sternebrae^g	Ribs
		Fore limb	Hind limb
Control	173	3 (1.7)	39 (22.5)	33 (19.1)	48 (27.7)	0	1 (0.6)	1 (0.6)	1 (0.6)^j
300	208	4 (1.9)	69 (33.2)	75 (36.1)	56 (26.9)	1 (0.5)	0	0	0
1,000	200	2 (1.0)	34 (17.0)	48 (24.0)	60 (30.0)	1 (0.5)	0	0	1 (0.5)^h
3,000	205	6 (2.9)	32 (15.6)	23 (11.2)	73 (35.6)	0	0	0	1 (0.5)^i
99 % confidence limits of the controls n= 170		0.2 – 6.31	15.15 – 32.21	12.19 – 28.29	19.22 – 37.3	0.00 – 3.07	0.00 – 4.29	0.00 – 4.29	0.00 – 4.29

 

Figures in parenthensis represent %.

a)     ossification

b)     proximal phalanges V

c)     proximal phalanges

d)     particularly ossification centres of the 5^thsternebrae still incompletely ossified (bipartite)

e)     centres of some throracic vertebrae still dumbbell-shaped

f)       centres of some throracic vertebrae bipartite

g)     centres displaced and irregularly ossified

h)     Basal fusion of left ribs 10 + 11

i)       Bifurcation of right rins no. 13

j)       Navy rib no. 13 (right)

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Treatment of dams was limited to gestation periods Day 6-15 of gestation only as opposed to a day before scheduled kill - certain maternal parameters not checked e.g corpora lutea was not determined.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(test substance administered on day 6-15 of gestation only)

Principles of method if other than guideline:

The test compound was administered orally to pregnant mice from day 6 until day 15 of gestation, inclusive. Dams were observed during the pregnancy and then killed on day 18 of the pregnancy and an autopsy performed to examine the dam and the foetuses.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): TK 10048
- Lot/batch No.: EN 26580 (1/75)

Species:

mouse

Strain:

NMRI

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Closed SPF breeding colony
- Weight at study initiation: 30 g
- Housing: Macrolon cages
- Diet: ad libitum; Nafag No. 890
- Water: ad libitum; tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.5
- Humidity (%): 56 ± 5
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

(2%; aqueous)

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Amount of vehicle (if gavage): 0.1mL/10g bw
- Lot/batch no. (if required): not stated
- Purity: not stated

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:4
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data but males of proven fertility were used.
- Verification of same strain and source of both sexes: yes, the albino mice were NMRI- derived and obtained from a closed SPF breeding colony
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 until day 15 of pregnancy inclusive.

Frequency of treatment:

once daily

Duration of test:

18 days

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not stated
- Rationale for animal assignment (if not random): not stated

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily

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, a mean food consumption graph was present in the study, but no details about individual animal food consumption.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No, as not a feeding study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 18
- Organs examined: Assessment of the dam's organs, especially the ovaries and uterus.

OTHER: The foetuses were subjected to careful external inspection and the condition of their body orifices was checked. They were then weighed individually followed by examination of the viscera and skeletal assessment .

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Not stated
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
-Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: No

Statistics:

Some statistics were present in the tables, although not in much detail. Standard deviation was only applied to weight of live foetuses. Confidence limits of the CMC-control for the skeletal assessment were present.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No reactions to treatment were observed. Body weight gain and food consumption were not reduced in a dose-related fashion.

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The foetuses were not adversely affected after the administration of either the low dose or the intermediate dose.

The ratios of implantation as well as embryolethality were comparable for all groups. The average weight of the foetuses from the experimental groups was not significantly diminished when compared with the vehicle control. There were few instances of malformed foetuses observed in the 300 mg/kg and 3,000 mg/kg dose group but these are considered to be of a spontaneous origin and not attributable to the administration of the compound. Cleft palate is known to be the most common malformation to occur spontaneously in the NMRI-derived mouse.

By applying the slicing technique, hypoplasia of lungs associated with dystopia cordis was detected in one out of 86 foetuses of the intermediate-dose group.

In the high dose group, a slightly increased number of still incompletely ossified sternebrae was seen in comparison to controls. This effect is considered by the authors to reflect an entirely non-specific sign of a slight delay of the physiological growth of the foetuses. However, because of the lack of historical control data verifying this claim, this effect can not be discounted. No teratogenic effects were noted. Mean body weights were higher in the high dose group than in the controls.

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

Table 1: Results of uterus examinations. 

	Dose groups (mg/kg bw)
	Historical controls (observed over 2 years)	Control	300	1,000	3,000
No. of dams	300	30	30	30	30
Spontaneous deaths	0	0	0	0	0
No of females with implantations	266	26	26	25	23
Mean No. of implantations	11.58	11.38	11.12	11.24	10.52
Embryonic resorptions (%)	6.2	7.4	10.4	7.5	5.0
Foetal resorptions (%)	1.1	1.7	0.7	0.7	1.6
Dead foetuses (%)	0.2	0.7	0	0.4	0.4
Live foetuses (%)	92.6	90.2	88.9	91.5	93
Live foetuses with malformations	9 / 2,887^a	0 / 267^d	3 / 257^b	0 /257	2 / 225^c
Mean weight of live foetuses (g)	1.16 ± 0.11	1.09 ± 0.14	1.11 ± 0.12	1.17 ± 0.13	1.17 ±0.13

 

(a)   6 cleft palates (1 assocoated with madibular hypoplasia), meningocele, 1 omphalocele + “kinky tail”, 1 kyphoscoliosis

(b)  2 cleft palates, I general oedema + bilat. Open eyes

(c)   2 cleft palates (1 in association with mandibular hypoplasia

(d)  99% confidence limits 0.00 – 1.92

Table 2: Skeletal changes in the foetuses

Dose group (mg/kg bw)	No. of skeletons examined	Phalangeal nuclei^a		Calcaneus^a	Supraoccipital bone^c	Sternebrae^d	Sternebrae^e
		Fore limb^b	Hind limb^b
Control	177	43 (24.3)	41 (23.2)	72 (40.7)	13 (7.3)	82 (46.3)	0
300	172	37 (21.5)	47 (27.3)	73 (42.4)	12 (6.9)	89 (51.7)	0
1,000	171	20 (11.7)	34 (19.9)	46 (26.9)	10 (5.8)	90 (52.6)	0
3,000	152	39 (25.7	46 (30.3)	58 (38.2)	16 (10.5)	90 (59.2)	0
99 % confidence limits of the controls n= 170		16.17- 32.96	15.69 – 32.35	30.71 – 49.86	3.06 – 13.60	36.51 – 55.98	0.00 – 2.90

 

Figures in parenthensis represent %.

a)     Ossification still present

b)     proximal phalanges (fore limb II – V only)

c)     incomplete ossification

d)     particularly ossification centres of the 5^thsternebrae still incompletely ossified (bipartite)

e)     whole sternum irregularly ossified

Conclusions:

The test substance did not exhibit any teratogenic or maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

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

Data on developmental toxicity is available from two species; from rat and mouse

 

In the first developmental toxicity study, the test substance (no data on purity) was administered to 25 pregnant Sprague-Dawley rats per dose by gavage at dose levels of 300, 1000, 3000 mg/kg bw/day in carboxymethyl cellulose (2%). Treatment was performed once daily from days 6 through 15 of gestation. Controls animals were treated with the vehicle only. Animals were sacrificed on day 21 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight, food consumption, or caesarean parameters in the treated dams. The maternal NOAEL is 3000 mg/kg bw/day. 

There were no treatment-related effects in developmental parameters. Implantation ratios and embryo lethality were comparable for all groups. The average body weights of foetuses in each group were not significantly different from that of controls. Minor anomalies (hypoplasia of the lungs, anasarca) occurred in similar incidences in the control, mid and high dose groups. These effects were equally represented in the historical control group. Based on these findings, no substance-related effect on embryonic or foetal development is assumed. The NOAEL for developmental toxicity is 3000 mg/kg bw/day.

Skeletal assessment did not reveal any clear-cut differences between the foetuses of the experimental group and those of the vehicle control. No teratogenic effects were noted. The NOAEL for teratogenicity is 3000 mg/kg bw/day (Ciba Geigy Ltd. 1976).

The developmental toxicity study in the rat is classified acceptable for assessment purposes of developmental toxicity as it satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rodents in all but 3 aspects. Firstly, unlike the current guideline, treatment of dams was limited to gestation periods Day 6-15 of gestation as opposed to treatment up to a day before scheduled kill. Secondly, not all caesarean parameters were evaluated e.g. corpora lutea were not determined. Finally, the test substance was not fully characterized in the study as no data on substance purity is given.

 

In the second developmental toxicity study, the test substance (no data on purity) was administered to 30 pregnant NMRI mice per dose by gavage at dose levels of 300, 1000, 3000 mg/kg bw/day in carboxymethyl cellulose (2%). Treatment was performed once daily from days 6 through 15 of gestation. Control animals were treated with the vehicle only. Animals were sacrificed on day 18 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight, food consumption, or caesarean parameters in the treated dams. The maternal NOAEL is 3000 mg/kg bw/day.

The ratios of implantation as well as embryo lethality were comparable for all groups. The average weight of the foetuses from the experimental groups was not significantly diminished when compared with that of control animals. In the high dose group, a slightly increased number of still incompletely ossified sternebrae was detected indicating a slight delay of the physiological growth of the foetuses. The LOAEL for developmental toxicity is 3000 mg/kg bw/day. The NOAEL for developmental toxicity is 1000 mg/kg bw/day.

The few instances of malformed foetuses observed in the 300 mg/kg and 3,000 mg/kg dose group were rather spontaneous in nature and not attributable to the administration of the compound. Cleft palate is known to be the most common malformation to occur spontaneously in the NMRI-derived mouse. No teratogenic effects were noted. The NOAEL for teratogenicity is 3000 mg/kg bw/day (Ciba Geigy Ltd., 1976).

The developmental toxicity study in the mouse is classified acceptable for assessment purposes of developmental toxicity as it satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rodents in all but 3 aspects. Firstly, unlike the current guideline, treatment of dams was limited to gestation periods Day 6-15 of gestation as opposed to treatment up to a day before scheduled kill. Secondly, not all caesarean parameters were evaluated e.g. corpora lutea were not determined. Finally, the test substance was not fully characterized in the study as no data on substance purity is given.

In the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD Test Guideline 422), described above, there were also no indications that the development of offsprings was impeded when parental animals were treated for 42 - 56 days with the test substance. On the basis of the observed parameters, a NOAEL for developmental toxicity of 1000 mg/kg bw/day was estimated (Nihon Bioresearch Center Inc., 2007).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available screening study for fertility/developmental toxicity and the two teratogencity studies are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.

 

During the four days covered in the screening study, no effects via lactation were observed.