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Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Extracts (petroleum), residual oil solvent
EC Number:
265-110-5
EC Name:
Extracts (petroleum), residual oil solvent
Cas Number:
64742-10-5
Molecular formula:
Not applicable
IUPAC Name:
Extracts (petroleum), residual oil solvent
Test material form:
liquid: viscous
Details on test material:
Concawe sample ref# S709

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 - 14 weeks old
- Weight at study initiation: 160 - 272 g (females at initiation of dosing), slightly above the taget weight of 180-230g
- Fasting period before study: N/A
- Housing: On arrival, females will be individually housed in Macrolon plastic cages (MIII type, height 18 cm). The cages will contain appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and will be equipped with water bottles.
- Diet (e.g. ad libitum): Free access to prepared pelleted diets, except during designated procedures. During the acclimatization period animals will have free access to standard powder rodent diet (SMR/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Municipal tap water will be freely available to each animal via water bottles.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean 19 - 20 deg C (target 18-24)
- Humidity (%): mean 45 - 52 % (target 40-70)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: 31 Mar 2021 To: 23 April 2021

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
The dietary route was selected as the test material is poorly soluble and difficult to administer by gavage on a repeated basis. In addition, dietary exposure where the animal is exposed throughout the day is thought to more closely mimic anticipated human dermal exposure (as opposed to a single large dose per day by gavage).

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared at least once weekly for use at room temperature for a maximum of
10 days.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used to prepare pelleted diets. The test item was weighed on a layer of powder feed to prevent it sticking to the container. Acetone (in a ratio of 35,8 mL per kg diet) was added in a stepwise manner to facilitate mixing of the test item with the powder diet and to prevent test item from sticking to the walls of the container. Then the bulk of the diet was added in a stepwise manner, while mixing. Water (approximately 15% in total) was added to aid pelleting.
- Storage temperature of food: The pellets were kept for approximately 24 hours at 35°C before final storage,
to dry the pellets and to evaporate the acetone. The control animals received similarly prepared pellets but without the test item. Diets will be kept at room temperature in the diet store room in the animal facility until use, if not used on the day of preparation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples were collected for analysis in Week 1 of dosing. Homogeneity samples taken for top and bottom dose groups only with homogeneity results averaged to provide concentration results for these groups. Concentration samples only were taken from the middle dose group.

Samples of approximately 60 g were taken from the diets. For determination of accuracy, samples were taken at the middle position (0% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the diets.

The concentrations analyzed in the diets of bottom, middle and top dose groups were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

The diets of bottom and top dose groups were homogeneous (i.e. coefficient of variation ≤ 10%).
Details on mating procedure:
Untreated females will be mated at the Supplier and will be at Day 0 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).
Duration of treatment / exposure:
Day 6 to Day 21 post-coitum
Frequency of treatment:
In diet
Duration of test:
Scheduled necropsy on Day 21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
Equivalent to 1350 ppm in diet, calculated mean intake 113 mg/kg/day
Dose / conc.:
300 mg/kg bw/day
Remarks:
Equivalent to 4100 ppm in diet, calculated mean intake 355 mg/kg/day
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Equivalent to 13000 ppm in diet, calculated mean intake 1102 mg/kg/day
No. of animals per sex per dose:
22 females per dose group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: selected on the basis of a range-finding study (6 animals per dose group; max dose 1000 mg/kg/day)
- Time of day for (rat) dam blood sampling and fasting period before blood sampling: Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Morbundity Checks at least twice daily throughout the study
Cage-side clinical observations at leadt once daily, starting on Day 6 post-coitum onwards up to the day prior to necropsy


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, animals were examined on days 2, 6, 13 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed on days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption of animals measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: regular basis throughout study by visual inspection of water bottles

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post-coitum day 21
- Organs examined: thyroid gland weight, macroscopic anomalies
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: The sex of each fetus based on anogenital distance, if possible
Blood sampling:
- Thyroid hormone analysis
- Volume collected 1.0 mL
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: Yes
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.

Parametric/Non-parametric
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis
test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

Incidence
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Indices:
Body Weight Gains: Calculated for the following intervals: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, and 6 to 21 post-coitum.
Corrected Body Weight Gain: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Overall Food Consumption: Calculated between each scheduled interval (individual data only) and as specified above for bodyweight gains. Summarization and statistical analysis intervals will reflect the same intervals as the bodyweight gains.
Test Item Intake: Calculated as concentration of test item in diet (ppm) against relative food consumption.
Pregnancy Rate (%): No. of pregnant females x 100 / No. of mated females
Male Fetuses (%): No. of male fetuses x 100 / No. of fetuses
Female Fetuses (%): No. of female fetuses x 100 / No. of fetuses
Pre-Implantation Loss (%): No. of corpora lutea – no. of implants x 100 / No. of corpora lutea
Post-Implantation Loss (%): No. of implants – no. of live fetuses x 100 / No. of implants
Litter % of Fetuses with Abnormalities: No. of fetuses in litter with a given finding x 100 / No. of fetuses in litter examined
Historical control data:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, erected fur was noted in five females (Nos. 68, 72, 79, 81 and 83) on 2-8 days (between Days 9-20 post-coitum), and hunched posture in two females (Nos. 83 and 88) on 3-4 days (between Days 9-18 post-coitum).

At 300 mg/kg/day, erected fur was noted in three females on 3-9 days between Days 13-21 post-coitum.

Other clinical signs noted during the treatment period (fur loss, flaking of skin and/or pale feces), occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On Day 9 post-coitum, mean body weight gain was decreased at 100 and 300 mg/kg/day (not statistically significant at 100 mg/kg/day) when compared to Day 6 post-coitum, and at 1000 mg/kg/day, a slight mean body weight loss of 2% was noted. As a result, mean body weight (gain) at 300 and 1000 mg/kg/day remained decreased (not always statistically significant) throughout the treatment period. On Day 21 post-coitum, body weights at 300 and 1000 mg/kg/day were 5 and 9% lower than control, respectively.
Mean gravid uterus weight was considered within normal range of biological variation. Mean body weight gain corrected for gravid uterus weight was lower at 300 and 1000 mg/kg/day (32 and 87% lower than control, respectively). At 1000 mg/kg/day, 6 out of 18 gravid females had body weight loss after correction for gravid uterus weight. At 300 mg/kg/day, no body weight loss was noted after correction for gravid uterus weight.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption of all treated groups was increased between Days 12-15 post coitum (up to 14% higher than control at 300 mg/kg/day). In absence of an effect on overall food consumption, and based on the direction of the change, this was considered not toxicologically relevant.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Of Note: Female Nos. 12 (control group), 67, 70, 81 and 88 (1000 mg/kg/day) were non-gravid. Female No. 62 (300 mg/kg/day) delivered its litter early on the day of scheduled necropsy. These females were therefore excluded from the summary data tables.

Mean total Triiodothyronine (T3) serum levels were lower at 1000 mg/kg/day (0.70x of control). However, as mean levels remained within the available historical control range, the observed reduction was considered not toxicologically relevant.

Mean total Thyroxine (T4) serum levels were lower at 1000 mg/kg/day (0.58x of control). Based on the magnitude of the effect and as T4 values in the high dose group were below the historical control range, a relation to treatment with the test item could not be excluded.

Mean serum levels of thyroid stimulating hormone (TSH) were higher at 1000 mg/kg/day (2.01x of control), although not reaching statistical significance. Based on the magnitude of the effect and as TSH values in the high dose group were above the historical control range, a relation to treatment with the test item could not be excluded.

However, the downstream biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not taken into account when determining the maternal NOAEL.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in thyroid gland weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations in the thyroid glands.

The recorded macroscopic finding of thyroid gland enlargement in one female at 1000 mg/kg/day had no microscopic correlate and was within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands.

The recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day, the pre-implantation loss was higher (12 vs. 5% in control, not statistically significant). This was mostly due to Female Nos. 52 and 63, with a low number of implantations. As the treatment with test item is started after implantation (Day 6 post-coitum), and in the absence of a dose-dependent relation, this was considered not related to treatment with the test item.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
Female No. 62 (300 mg/kg/day) delivered its litter early on the day of scheduled necropsy. The number of females with viable litters for evaluation was 21, 22, 21 and 18 in the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled necropsy, Female Nos. 12 (control group), 67, 70, 81 and 88 (1000 mg/kg/day) were non-gravid. Salewski staining of the uterus of these females showed no implantation sites, indicating that there has been no loss of pregnancy. Therefore, these cases of non-pregnancy were considered a chance finding and unrelated to treatment with the test item.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
body weight and weight gain
Remarks on result:
other: report
Remarks:
Value from study report
Key result
Dose descriptor:
NOAEL
Effect level:
113 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: review
Remarks:
Value from independent review

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetal body weights were decreased for male (5.7%) and female fetuses (7.2%) at 1000 mg/kg/day compared to control, as well as combined fetal body weights (6.5%). Values were below the lower limit of the historical control range.
Mean fetal body weights at 300 and 100 mg/kg/day were considered unaffected by treatment with the test item.

It cannot be ruled out that this decrease was secondary to the observed maternal toxicity at 1000 mg/kg/day. As the low fetal body weights were observed in the absence of retarded growth for ossification parameters, this was considered to be non-adverse.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day, the number of live fetuses was slightly reduced (0.88x of control) compared to control (not statistically significant). This was mostly due to Female Nos. 52 and 63, with a low number of implantations. In the absence of a dose-dependent relation, this was considered not related to treatment with the test item.
The litter size at 100 and 1000 mg/kg/day were considered within normal range of biological variation.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day, the number of live fetuses was slightly reduced (0.88x of control) compared to control (not statistically significant). This was mostly due to Female Nos. 52 and 63, with a low number of implantations. In the absence of a dose-dependent relation, this was considered not related to treatment with the test item.
The litter size at 100 and 1000 mg/kg/day were considered within normal range of biological variation.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The mean fetal AGD for males was decreased at 300 and 1000 mg/kg/day (0.95 and 0.94x of control, respectively). This was considered to be secondary to the observed lower fetal weights, and not a direct effect of treatment with the test item, since the mean normalized fetal AGD was not affected.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Three external malformations were observed in this study, one in the control group and two in the high dose group. In the high-dose group, omphalocele (organs sticking out) and malrotated hindlimbs were observed in fetuses 71-L2 and 77-R10, respectively. These malformations were considered chance findings due to their single occurrence and/or observation in the control group. The control fetus (No. 22-L3) presented a proboscis (elongated nose).
There were no external variations observed in any groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were observed in 3 (3), 2 (2), and 5 (4) fetuses (litters) from the control, low- and high-dose groups, respectively and affected the forelimb, ribs, skull, sternebrae and vertebrae. No malformations were found in the mid-dose group.
Of the skeletal malformations discovered, several were underlying to the external finding of fetus 22-L3 presenting with proboscis. All other malformations occurred incidentally and/or in the control group and therefore, not deemed test-item related.
Of the skeletal variations observed the most noteworthy was that the incidence of wavy ribs was significantly lower in the mid- and high-dose groups when compared to the control group. The lower incidence for this finding is considered to not have any detrimental effect.
Therefore, the notable group distribution of bent ribs is considered to have occurred by chance and was not related to treatment with the test item.
All other skeletal variations occurred in either isolated cases, in the absence of a dose relationship and/or in the control group and hence considered not test-item related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no visceral malformations observed in this study.
Visceral variations that were noted principally affected the liver, with supernumerary lobes observed across all groups. However, with no dose dependent effect and including observations in the control group, this was considered not related to treatment with the test item. Isolated variations in the spleen and ureter were noted, each affecting one fetus and hence considered not test item-related.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No treatment related developmental effects noted
Remarks on result:
other: report
Remarks:
Value from study report
Key result
Dose descriptor:
NOAEL
Effect level:
355 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: review
Remarks:
Value from independent review

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 102 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Applicant's summary and conclusion

Conclusions:
Based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the following No Observed Adverse Effect Levels (NOAELs) for Residual Aromatic Extract (CAS number 64742-10-5) were established:

Maternal: 100 mg/kg/day (1350 ppm, actual intake of 113 mg/kg/day)

Developmental: 300 mg/kg/day (4100 ppm, actual intake of 355 mg/kg/day)
Executive summary:

Time-mated female Wistar Han rats were treated with Residual Aromatic Extract (CAS number 64742-10-5) from Day 6 to 21 post-coitum, inclusive by dietary administration at target dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received only the standard powder rodent diet.

 

Maternal findings

No mortality occurred during the treatment period.

 

At 1000 mg/kg/day, body weight loss was noted at the start of treatment with the test item. Mean body weight (gain) remained decreased (not always statistically significant) throughout the treatment period. Mean body weight gain corrected for gravid uterus weight was lower compared to control. Together with erected fur and hunched posture that were noted for some females, these findings were considered adverse.

 

At 300 mg/kg/day, erected fur was noted for some individual females, mean body weight (gain) and mean body weight gain corrected for gravid uterus weight were slightly lower compared to control. However, as the reduction in body weight gain was not entirely correlated with reduced food consumption, and comparison of body weight gain with controls between gestation days 6 and 21 shows that the treated group had not reached parity with control), the NOAEL for maternal toxicity is conservatively set at the mid-dose group (113 mg/kg/day).

 

Reduced mean serum level of total T4 was noted at 1000 mg/kg/day, that was below the available historical control range. Based on the magnitude of the effect and as values were below the lower limit of the historical control range, this decrease was considered test item related.

Increased mean serum level of TSH was noted at 1000 mg/kg/day, although not reaching statistical significance. Based on the magnitude of the effect and as values were above the upper limit of the historical control range, this increase was considered test item related.

However, the downstream biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not taken into account when determining the maternal NOAEL.

 

No test item-related toxicologically relevant effects were noted in any of the remaining maternal parameters investigated in this study

 

Developmental findings

At 1000 mg/kg/day, lower fetal weights were observed compared to control. Fetal body weights have a small variation in general and given the magnitude of the effect (6-7%), and as mean values at 1000 mg/kg/day were below the lower limit of the historical control range, these findings were considered test item-related. It cannot be ruled out that this decrease was secondary to the observed maternal toxicity at 1000 mg/kg/day. However, as this reduction was statistically significant, the NOAEL is conservatively set at the mid-dose level (300 mg/kg/day targeted; 355 mg/kg/day actual dose).

 

No toxicologically relevant changes were noted in any of the remaining developmental parameters investigated in this study