Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In accordance with column 1 of REACH Annex IX, the reproductive toxicity study (as required in section 8.7.3.) does not need to be conducted as the data from the 28-day and 90-day repeated dose toxicity studies as well as the developmental toxicity study do not indicate adverse effects on reproductive organs or tissues. Additionally, exposure of this substance to humans is expected to be limited.

Link to relevant study records

Reference

Endpoint:

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

Data waiving:

exposure considerations

Justification for data waiving:

the study does not need to be conducted because relevant human exposure can be excluded as demonstrated in the relevant exposure assessment

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No effects on the gonads in the repeated dose studies and developmental toxicity available were noted.

The delayed ossification effect in the developmental toxicity study is considered to be a secondary effect of the reduced maternal body weight during day 6 -9 of gestation. No direct developmental toxicity was observed.

Considering the above mentioned data, and the low NOAEL in the 90-day tox study (32 mg/kg bw/d), possible reproduction effects are expected to occur only at maternal toxicity levels. Therefore, the repeated dose toxicity level stays critical for DNEL derivation. Therefore a 1 gen reproduction study is not considered necessary, to provide additional information.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study (OECD 414) in New Zealand White rabbits, the No Observed Adverse Effect Level (NOAEL) for LOWINOX® CPL was established as being 50 mg/kg. The developmental NOAEL was established as being 15 mg/kg, based on the increased incidence of fused sternebrae (malformation) at 50 and 150 mg/kg.

In a further prenatal developmental toxicity study in rats, the substance administered by gavage during major organogenesis in rats resulted in no indication of teratogenicity, but did result in increased incidences of common fetal skeletal variations at 1000, 2000 and 3000 mg/kg/day. The NOAEL for maternal toxicity was 1000 mg/kg/day and a NOAEL for developmental toxicity of 1000 mg/kg/day was established.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 April 2016 to 15 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See under Overall Remarks below

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test System:
Rabbit: female albino rabbits, New Zealand White (NZW) strain (SPF-Quality), from a non-inbred laboratory colony. Nulliparous, non-pregnant and untreated females were used at initiation of the study.
Stock male NZW rabbits were used for mating with the females. These males were adult and proven fertile. After mating these males were placed back in their stock for possible use in future studies.
Rationale:
This species and strain of rabbit has been recognized as appropriate for developmental toxicity studies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Source: Charles River, Chatillon sur Chalaronne, France.
Number of animals: F0-generation: 88 females.
F1-generation: 769 fetuses.
Age at delivery: Females were approximately 17-19 weeks
Acclimatization: At least 5 days prior to pairing.
Health inspection: Upon receipt of the animals.
Randomization: The animals were allocated to the groups by computer-generated random algorithm according to body weight, with all animals within ± 20% of the mean. Upon observation of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
Identification: By tattoo in the ear.
Mating procedures: One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Veterinary examination: No veterinary inspection of the rabbits was performed during the experimental phase of this study

Animal Husbandry
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The photoperiod is between 07.00 and 19.00 hrs dailyt. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Females were individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy). In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were provided during the study period.
Water: Free access to tap-water.
Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

other: 1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose: BUFA, IJsselstein, The Netherlands; water: Elix, Millipore S.A.S., Molsheim, France) with 0.1% Tween-80 (Merck-Schuchardt, Hohenbrunn, Germany).

Details on exposure:

Rationale for vehicle: Based on trial formulations performed at Charles River Den Bosch.
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item.
Appearance of formulations: Suspension (Groups 2-4)
Storage conditions: At room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (22 June 2016), according to a validated method (Test Facility Study No. 512431). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Analytical conditions:
Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Acquity UPLC TUV detector (Waters)
Column Acquity UPLC BEH C18, 50 mm +/- 2.1 mm i.d., dp = 1.7 μm (Waters)
Column temperature 40°C +/- 1°C
Injection volume 5 μL
Mobile phase A - methanol, B - water
Gradient:
Time = 0 mins: 80% A, 20% B
Time = 5 mins: 100% A, 0% B
Time = 7 mins: 100% A, 0% B
Time = 7.1 mins: 80% A, 20% B
Time = 11 mins: 80% A, 20% B
Flow 0.5 mL/min
UV detection 283 nm

Preparation of solutions
Stock and spiking solutions
Stock and spiking solutions of the test item were prepared in methanol at a concentration of 1000 mg/L. In order to dissolve the test item the solutions were ultrasonicated for 15 minutes.
Calibration solutions
Five calibration solutions in the concentration range of 0.8 – 50 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was methanol.
Quality control (QC) samples
Approximately 500 mg blank vehicle was spiked with the test item at a target concentration of 1 or 30 mg/g. The QC samples were treated similarly as the study samples

Results:
In the Group 1 formulation, no test item was detected.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
Homogeneity
The formulation of Group 2 was homogeneous (i.e. coefficient of variation ≤ 10%). A relatively high coefficient of variation (10%) was noted for the results of the Group 4 formulation. During sampling and analysis no observations were made which could have explained this relatively high variation. As the result complied with its specification (i.e. coefficient of variation ≤ 10 %), no further analyses were required.

Details on mating procedure:

One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

From Days 6 to 28 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 female rabbits per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Oral gavage, using a plastic catheter attached to a plastic disposable syringe. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Rationale for Dose Levels
Dose levels were selected based on the results of the dose range finding study.Treatment at 300, 600 and 1000 mg/kg resulted in severe toxicity. Almost all treated animals showed severe body weight loss (up to 13%), reduced food consumption and reduced production of (pale) faces. In addition, lean appearance was noted in 4/6 females at 600 mg/kg and 5/6 females at 1000 mg/kg. Other treatment-related clinical signs at 600 and 1000 mg/kg included pale appearance, hunched posture and piloerection. Based on these findings, it was decided in consultation with the Sponsor to sacrifice all Groups 2-4 animals for animal welfare reasons. To determine the highest tolerated dose level for the main study, two additional treatment groups of 50 and 150 mg/kg were added. At 50 mg/kg, no toxicologically relevant findings were observed. At 150 mg/kg, three females were non-pregnant. As no dose-response relation was observed, this was not considered to be treatment related. Food consumption was slightly reduced at 150 mg/kg on Days 13-16, but this was recovered the days after. All pregnant females had live fetuses. No effects on pre- and post-implantation loss and fetal weights were observed. External examination resulted in one malformation, carpal flexure, observed in one fetus at 50 mg/kg. According to these results, the highest tolerated dose was established as being 150 mg/kg.

Maternal examinations:

Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect abortion or premature birth, if applicable.
Body weights: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.
Food consumption: Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Necropsy
All animals surviving to the end of the observation period and all moribund animals were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol®20%; AST Farma B.V., Oudewater, The Netherlands) and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.
All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Necropsy was conducted on the following days:
Females surviving to planned necropsy: Day 29 post-coitum.
Euthanized in extremis (female no. 78): On Day 20 post-coitum, when pain, distress or discomfort was considered not transient in nature or was likely to become more severe.
Each ovary and uterine horn of all animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
The number of corpora lutea.
The weight of the (gravid) uterus.
The number and distribution of live and dead fetuses.
The number and distribution of embryo-fetal deaths.
The weight of each fetus.
The sex of each fetus (during further fetal examination).
Externally visible macroscopic fetal abnormalities.
Animal no. 78 (Group 4), which was sacrificed before planned necropsy, was subjected to relevant examinations of the ovaries and uterine horns. Findings were reported in the individual data tables only.

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

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
External
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (=60mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.
Visceral (Internal)
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.
Malformations were collected and fixed in 10% buffered formalin, if possible.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands) for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.
Skeletal
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (Ref 4). Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. As possible treatment related effects were observed in the high dose group, skeletal examination was extended to all fetuses from the low and mid dose group.
All specimens were archived in glycerin (BRENNTAG Nederland B.V., Dordrecht, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

The following statistical methods were used to analyze the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
The Steel-test ((many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites)/number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses)/number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%) = number of viable fetuses affected/litter/number of viable fetuses/litter x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals at 150 mg/kg showed a slightly higher incidence and severity of reduced faeces production compared to control animals (20 animals at 150 mg/kg, vs. 16 in the control group, and 17 animals each at 15 and 50 mg/kg). The incidence of reduced faeces production at 15 and 50 mg/kg was considered unrelated to treatment since the incidence was comparable with control levels, and since this finding is regularly seen for rabbits of this age and strain housed and treated under the conditions in this study. It was ascribed to the large variation in food consumption observed among individual animals of these groups.
A lean appearance was noted for four animals at 150 mg/kg during the last 3-9 days of treatment (excl. female no. 78 that died preterm; see previous section 7.2.1). Also one female each in the control and 50 mg/kg groups had a lean appearance on the last two days of treatment. At the isolated incidence and in the absence of a dose-response, it was considered not related to treatment with the test item.
Clonic spasms were noted for one female (no. 70) at 150 mg/kg on the morning of Day 10 post-coitum before treatment. After the female was recovered, she was dosed. No further seizures were seen. This type of spasms is sometimes seen for rabbits. Based on its isolated incidence and the fact that occurred just before dosing, it was considered unrelated to treatment.
For one female at 50 mg/kg (no. 66) red fluid on the manure tray was observed on Days 27 and 28 post-coitum. Since she had a normal litter, no toxicological relevance was attached to this finding.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. These signs did not show any apparent dose-related trend and/or were also observed during the pre-treatment period. At the incidence observed, these signs were considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One female at 150 mg/kg (no. 78) had to be euthanized on Day 20 post-coitum due to unexpected high toxicity in this female. From start of treatment (Day 6 post-coitum) onwards, the animal had a remarkably low intake of food and water (taken from study daybook), resulting in reduced faeces production (at a slight to severe degree) from Day 9 post-coitum onwards. Body weight loss was observed from Day 9 post-coitum onwards, with a total loss of 13% from Day 6 to Day 20 post-coitum. Correlating in-life and necropsy observations were a lean appearance and emaciation on Day 20 post-coitum. There were no other necropsy findings.
All remaining females survived until scheduled necropsy on Day 29 post-coitum.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For the 150 mg/kg group no changes in body weight were noted from Days 9-16 post-coitum, followed by 2% body weight loss from Day 16-20 post-coitum. Subsequently, the rate in body weight gain was similar to or slightly higher than that of the other groups. However, due to the retarded growth and body weight loss seen during the first 14 days of treatment at the high dose level, mean values for body weights remained below control values until the end of the observation period. Compared to the concurrent control group, mean body weight and body weight gains were statistically significantly lower on Day 20 post-coitum and from Days 13-23 post-coitum, respectively.
After correction for gravid uterus weight, there was a trend towards slightly lower body weight gain in the high dose group. This was caused mainly by female nos. 70 and 85 (both noted lean from Day 26 or Day 23 post-coitum onwards), who had a body weight loss of 20% and 24.2%, respectively, after correction.
Mean body weight and body weight gain (before and after correction for gravid uterus weight) of females at 15 and 50 mg/kg remained in the same range as controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 150 mg/kg group, reduced food consumption (absolute and relative to body weight) was noted from Days 9-16 post-coitum, followed by progressive recovery until the end of the observation period. When compared to the concurrent control group, mean values were clearly lower from Days 9-23 post-coitum (not statistically significant for relative food intake from Days 20-23 post-coitum), whereas higher values were recorded from Days 23-29 post-coitum (reaching statistically significance from Days 23-26 post-coitum). This latter finding was considered to have arisen as a result of the relatively low mean food intake in the control group over these days.
A similar trend was noted for the 50 mg/kg group, but changes were much less pronounced and did not reach statistical significance when compared to the concurrent control group. Therefore, no toxicologically relevance was attached to this finding.
At 15 mg/kg, mean food consumption (both absolute and relative) remained in the same range as controls.
In general, relatively large variations in food consumption occurred among individual animals of all groups (including controls). This is more often seen for rabbits of this age and strain which are housed and treated under the conditions in this study.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
One female (no. 39) in the low dose group had its left uterus horn interrupted. This was a congenital abnormality and unrelated to treatment that started after the animal had reached adolescence.
The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show any apparent dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of pre- or post-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre- or post-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of pregnant females, corpora lutea and implantation sites, and pre- or post-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Dead fetuses:

no effects observed

Description (incidence and severity):

One dead fetus was noted in litter 27 of Group 2.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

In the control, 15, 50 and 150 mg/kg groups, respectively 2, 1, 1 and 0 females were non-pregnant. All remaining females were pregnant.

Details on maternal toxic effects:

Clear toxicity was observed at 150 mg/kg.
One female at 150 mg/kg (no. 78) had to be euthanized on Day 20 post-coitum due to unexpected high toxicity in this female. From start of treatment (Day 6 post-coitum) onwards, this animal had a remarkably low intake of food and water (taken from study daybook), resulting in reduced faeces production (at a slight to severe degree) from Day 9 post-coitum onwards. Body weight loss was observed from Day 9 post-coitum onwards, with a total loss of 13% from Day 6 to Day 20 post-coitum. Correlating in-life and necropsy observations were a lean appearance and emaciation on Day 20 post-coitum.
In general, animals at 150 mg/kg showed a slightly higher incidence and severity of reduced faeces production compared to control animals (20 animals at 150 mg/kg, vs. 16 in the control group, and 17 animals each at 15 and 50 mg/kg). A lean appearance was noted for four animals at 150 mg/kg during the last 3-9 days of treatment (excl. female no. 78 that died preterm; see previous section). These findings were in line with the (on average) absence of weight increase or weight loss and lower food consumption throughout the treatment period.
No maternal toxicity was observed in the 15 and 50 mg/kg groups.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly lower mean male and female fetal body weights were noted in the treated groups, compared to controls. As changes were only slight and reached no statistical significance, were within the range of available historical control data and no dose-related response could be established, no toxicological relevance was attached to this finding.
Mean combined (male and female) fetal body weights were 40.2, 37.0, 38.4 and 36.9 grams for the control, 15, 50 and 150 mg/kg groups, respectively.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Slightly lower mean male and female fetal body weights were noted in the treated groups, compared to controls. As changes were only slight and reached no statistical significance, were within the range of available historical control data and no dose-related response could be established, no toxicological relevance was attached to this finding.
Mean combined (male and female) fetal body weights were 40.2, 37.0, 38.4 and 36.9 grams for the control, 15, 50 and 150 mg/kg groups, respectively.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

One dead fetus was noted in litter 27 of Group 2

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 150 mg/kg.
Mean sex ratios (males:females) were 49:51, 45:55, 52:48 and 51:49 for the control, 15, 50 and 150 mg/kg groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size in any group.
Mean litter sizes were 9.2, 10.0, 8.8 and 9.1 viable fetuses/litter for the control, 15, 50 and 150 mg/kg groups, respectively.

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 150 mg/kg.
The only two external malformations of this study were observed in Group 2. These were a cleft palate (entire length) in fetus A025-09 and a distended abdomen in fetus A026-01 that was viscerally confirmed by the presence of watery-clear ascites. The single occurrence of these malformations at the low dose does not indicate a relation to treatment and therefore both malformations were considered chance findings.
There were no external variations seen for any fetus in any group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

As eight fetuses out of five litters at the high dose had fused sternebrae versus none in the control group, it was decided to extend the skeletal examinations to Groups 2 and 3.
There was a dose-related increased incidence of fetuses with fused sternebrae at 50 and 150 mg/kg. This skeletal malformation was observed in 0 (0), 1 (1), 3 (3) and 8 (5) fetuses (litters) at an incidence of 0.0 %, 0.4%, 1.5% and 4.6% per litter in Groups 1, 2, 3, and 4, respectively. The incidence in Group 4 was higher than the historical control maximum value (2.4% per litter) and given the observed dose response and fact that the Group 3 incidence was above the 95th percentile of the available historical control data of the Test Facility (1.3% per litter), also the occurrence of fused sternebrae in Group 3 was considered to be adverse. As in Group 2 only one litter was affected and the incidence was below the mean value of the available historical control data (1.0% per litter), the occurrence in Group 2 was considered to be a chance finding.
Another skeletal malformation affecting the sternum, sternal anomaly, occurred in one fetus each of Groups 2 and 3 (A025-09 and A066-07, respectively). These anomalies were not grouped with the fused sternebrae as the aberrations were more complex. Moreover, both fetuses had external, visceral and/or skeletal malformations as well (A025-09 had cleft palate, diaphragmatic hernia, bent femurs and anomalous ribs, and A066-07 had two cardiovascular abnormalities). Therefore, and because they occurred singly, they were not considered to be treatment related.
Remaining skeletal malformations occurred at a very low incidence and as these were observed in either Group 2 or Group 3 only, they were not considered to be treatment related.
Skeletal variations occurred at an incidence of 77.8%, 79.8%, 81.9% and 70.8% per litter in Groups 1, 2, 3 and 4, respectively.
Noteworthy is the variation of slightly to moderately malaligned sternebrae for which the incidence in Group 2 was statistically significantly higher than the control value. Mean litter incidences of this finding were 12.7%, 20.0%, 14.3% and 10.3% per litter in Groups 1, 2, 3 and 4, respectively. For unknown reason, all these numbers were higher than the historical control maximum value (9.9% per litter). However, since no dose response could be established, the higher number of slightly to moderately malaligned sternebrae in Group 2 was considered not toxicologically relevant and not treatment related.
Other skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence trend, at frequencies that were within the range of available historical control data or were observed in a control fetus only.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 150 mg/kg.
Visceral malformations occurred in 2 (2), 8 (7), 1 (1) and 2 (2) fetuses (litters) in Groups 1, 2, 3, and 4, respectively. Absence of a lung lobe was the most common malformation that was observed in four Group 2 fetuses and one fetus each in Groups 1 and 4. As a dose response for this finding could not be established and as it is also the most common visceral malformation in historical controls, it was not considered to be treatment related.
The other visceral malformations occurred singly in one or two groups and therefore were considered to be chance findings.
All the variations noted were considered not to be treatment related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data or were observed in a control fetus only.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Tables are attached below under background material.

Conclusions:

Mated female Zealand White rabbits were assigned to four dose groups, each containing twenty-two animals. The test item was administered once daily by oral gavage from Day 6 to 28 post-coitum, inclusive,at doses of 15, 50 and 150 mg/kg (Groups 2, 3 and 4, respectively). The rabbits of the control group (Group 1) received the vehicle,1% Aqueous carboxymethyl cellulose with 0.1% Tween-80, alone.
Accuracy and homogeneity of formulations were demonstrated by analyses.
Maternal findings
Clear toxicity was observed at 150 mg/kg.
One female at 150 mg/kg (no. 78) had to be euthanized on Day 20 post-coitum due to unexpected high toxicity in this female.From start of treatment (Day 6 post-coitum) onwards, this animal had a remarkably low intake of food and water (taken from study daybook), resulting in reduced faeces production (at a slight to severe degree) from Day 9 post-coitum onwards.Body weight loss was observed from Day 9 post-coitum onwards,with a total loss of 13% from Day 6 to Day 20 post-coitum. Correlating in-life and necropsy observations were a lean appearance and emaciation on Day 20 post-coitum.
In general, animals at 150 mg/kg showed a slightly higher incidence and severity of reduced faeces production compared to control animals (20 animals at 150 mg/kg, vs. 16 in the control group, and 17 animals each at 15 and 50 mg/kg). A lean appearance was noted for four animals at 150 mg/kg during the last 3-9 days of treatment (excl. female no. 78 that died preterm; see previous section). These findings were in line with the (on average) absence of weight increase or weight loss and lower food consumption throughout the treatment period.
No maternal toxicity was observed in the 15 and 50 mg/kg groups.
Developmental findings
There was a dose-related increased incidence of fetuses with fused sternebrae at 50 and 150 mg/kg. This skeletal malformation was observed in 0 (0), 1 (1), 3 (3) and 8 (5) fetuses (litters) at an incidence of 0.0 %, 0.4%, 1.5% and 4.6% per litter in Groups 1, 2, 3, and 4, respectively. The incidence in Group 4 was higher than the historical control maximum value (2.4% per litter) and given the observed dose response and fact that the Group 3 incidence was above the 95th percentile of the available historical control data of the Test Facility (1.3% per litter), also the occurrence of fused sternebrae in Group 3 was considered to be adverse.
No toxicologically relevant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external and visceral malformations and variations, and skeletal variations).
In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for LOWINOX® CPL was established as being 50 mg/kg. The developmental NOAEL was established as being 15 mg/kg, based on the increased incidence of fused sternebrae (malformation) at 50 and 150 mg/kg.

Executive summary:

Title

Prenatal developmental toxicity study of LOWINOX® CPL in rabbits by oral gavage.

Guidelines

The study procedures described in this report were based on the following guidelines:

Organization of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

 Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.31: "Prenatal Developmental Toxicity Study". Official Journal of the European Union No. L142, May 2008.

The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

Rationale for dose levels

Dose levels were selected based on the results of the dose range finding study (Test Facility Study No. 512426). Treatment at 300, 600 and 1000 mg/kg resulted in severe toxicity. Almost all treated animals showed severe body weight loss (up to 13%), reduced food consumption and reduced production of (pale) faeces. In addition, lean appearance was noted in 4/6 females at 600 mg/kg and 5/6 females at 1000 mg/kg. Other treatment-related clinical signs at 600 and 1000 mg/kg included pale appearance, hunched posture and piloerection. Based on these findings, it was decided in consultation with the Sponsor to sacrifice all Groups 2-4 animals for animal welfare reasons. To determine the highest tolerated dose level for the main study, two additional treatment groups of 50 and 150 mg/kg were added. At 50 mg/kg, no toxicologically relevant findings were observed. At 150 mg/kg, three females were non-pregnant. As no dose-response relation was observed, this was not considered to be treatment related. Food consumption was slightly reduced at 150 mg/kg on Days 13-16, but this was recovered the days after. All pregnant females had live fetuses. No effects on pre- and post-implantation loss and fetal weights were observed. External examination resulted in one malformation, carpal flexure, observed in one fetus at 50 mg/kg. According to these results, the highest tolerated dose was established as being 150 mg/kg.

Study outline

Eighty-eight mated female New Zealand White rabbits were assigned to four groups of 22 animals each. The test item, LOWINOX® CPL, was administered once daily by oral gavage from Days 6 to 28 post-coitum, inclusive, at doses of 15, 50 and 150 mg/kg Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle,1% Aqueous carboxymethyl cellulose with 0.1% Tween-80, alone.Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. The fetuses of all groups were stained with Alizarin Red S, followed by skeletal examinations.

RESULTS

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings

Clear toxicity was observed at 150 mg/kg.

One high dose female was killed in extremis on Day 20 post-coitum.From start of treatment (Day 6 post-coitum onwards), this animal had a remarkably low intake of food and water (taken from study daybook), resulting in reduced faeces production and body weight loss from Day 9 post-coitum onwards, with a total loss of 13% from Day 6 to Day 20 post-coitum. Correlating in-life and necropsy observations were a lean appearance and emaciation on Day 20 post-coitum.

In general, animals at 150 mg/kg showed a slightly higher incidence and severity of reduced faeces production compared to control animals (20 animals at 150 mg/kg, vs. 16 in the control group, and 17 animals each at 15 and 50 mg/kg). A lean appearance was noted for four animals at 150 mg/kg during the last 3-9 days of treatment (excl. female no. 78 that died preterm; see previous section). These findings were in line with the (on average) absence of weight increase or weight loss and lower food consumption throughout the treatment period.

No maternal toxicity was observed in the 15 and 50 mg/kg groups.

Developmental findings

There was a dose-related increased incidence of fetuses with fused sternebrae at 50 and 150 mg/kg. This skeletal malformation was observed in 0 (0), 1 (1), 3 (3) and 8 (5) fetuses (litters) at an incidence of 0.0 %, 0.4%, 1.5% and 4.6% per litter in Groups 1, 2, 3, and 4, respectively. The incidence in Group 4 was higher than the historical control maximum value (2.4% per litter) and given the observed dose response and fact that the Group 3 incidence was above the 95^thpercentile of the available historical control data of the Test Facility (1.3% per litter, also the occurrence of fused sternebrae in Group 3 was considered to be adverse.

 

No toxicologically relevant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external and visceral malformations and variations, and skeletal variations).

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for LOWINOX® CPL was established as being 50 mg/kg. The developmental NOAEL was established as being 15 mg/kg, based on the increased incidence of fused sternebrae (malformation) at 50 and 150 mg/kg.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 October 1996 - 30 December 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: TSCA (1985), OPPTS (1996), Japanese MITI (1993) and OECD 414 (1981)

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. Raleigh, NC
- Age at study initiation: 10 weeks on gestational day 0
- Weight at study initiation: 215.8 – 274.4 g on gestational day 0
- Fasting period before study: no
- Housing: singly in solid bottom polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64 – 79 ◦F (18 - 26 ◦C)
- Humidity (%): 30 - 70
- Air changes (per hr): not described
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 21, 1996 To: November 20, 1996

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): once
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: 60-70 ◦F (16 -21◦C)

VEHICLE
- Justification for use and choice of vehicle (if other than water): not provided
- Concentration in vehicle: 0, 100, 200, and 300 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg body weight
- Lot/batch no. (if required): 211093 (test compound)
- Purity: 100% (test compound)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations were homogeneous and stable for at least 25 days. Dosing formulations with assay values of 90-110% of target were considered suitable for use.

Analysis of Wingstay L in corn oil: Appendix I of main report, RTI-65C-6503
Waters 845 HPLC system
Columns (2 in series): Phenomenex Phenogel 5 (300 mm x 7.8 mm i.d.), 100 Angstrom pore size
mobile phase: 100% THF (isocratic)
flow-rate: 0.7 ml/min
detection: UV at 285 nm
injection solvent: methanol
injection volume: 10 uL

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1:1
- Length of cohabitation: until there were 25 sperm-positive females per group
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Such a procedure was not adopted
- Further matings after two unsuccessful attempts: not applicable
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none

Duration of treatment / exposure:

Gestational days 6 through 19

Frequency of treatment:

once daily

Duration of test:

Sacrifice on gestational day 20.

Remarks:

Doses / Concentrations:
0, 1000, 2000 or 3000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 sperm-positive females per group.

Control animals:

yes

Details on study design:

- Dose selection rationale: based on two preliminary dose-range finding studies
- Rationale for animal assignment (if not random): not applicable
- Other: not applicable

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data
- Time schedule: not applicable
- Cage side observations checked in table [No.?] were included. not applicable

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily during dosing period, once daily at other time intervals

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 15, 18, 19, 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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: not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: not applicable

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: liver, ovaries and uterus

OTHER: none

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: none

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

yes

Indices:

none

Historical control data:

yes

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No dams died, aborted or delivered early. Maternal weight gain was significantly reduced with a dose-response at all doses for gestational days 6-9. Maternal gestational weight gain was significantly reduced at 3000 mg/kg/day. Maternal absolute and relative liver weights were significantly increased at all doses. Maternal feed consumption was significantly reduced at 3000 mg/kg/day for gestational days 6-9.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on any gestational parameters, including pre- or post-implantation loss, number of live fetuses per litter, fetal sex ratio (% males per litter) or fetal body weight per litter. There were no treatment-related changes in the incidence of pooled external, visceral (including craniofacial), skeletal or total fetal malformations. Percent fetuses with variations per litter showed a dose-related significant increase at all doses, only when sexes were combined, due to treatment-related increases in the incidence of two common fetal skeletal variations: rudimentary rib on lumbar I (bilateral, right or left) and reduced ossification in thoracic centra (normal cartilage, bipartite ossification center and dumbbell cartilage, bipartite ossification centre).

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skeletal variations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

none

Conclusions:

Wingstay L administered by gavage during major organogenesis in rats resulted in no indication of teratogenicity, but did result in increased incidences of common fetal skeletal variations at 1000, 2000 and 3000 mg/kg/day. The NOAEL for maternal toxicity was 1000 mg/kg/day based on decreased body weight change during day 6-9 and increased liver weights and a NOAEL for developmental toxicity of 1000 mg/kg/day was established.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Klimisch 1 study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is classified as Repr. 2: H361 in accordance with the CLP Regulation.