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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jul 2017 to 29 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(4-isobutyl-2-methylphenyl)propanal
EC Number:
811-285-3
Cas Number:
1637294-12-2
Molecular formula:
C14H20O
IUPAC Name:
3-(4-isobutyl-2-methylphenyl)propanal
Test material form:
liquid
Specific details on test material used for the study:
Name (as stated in the report): Nympheal
Lot No: SC00019702
Aspect: Colourless to pale yellow liquid
Expiration date: 26 February 2018

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.-
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Age at study initiation: 11 wks old (males) and 14 wks old (females)
- Weight at study initiation: males weighed 289 and 325 g and females weighed between 208 and 245 g
- Environmental Acclimation: 6 days prior to start of the pretest period (females) or commencement of exposure (males). The females were acclimatized to the jackets at least 5 days prior to and during pretest. The males were acclimatized to the jackets according to standard procedures.
- Housing: On arrival and following the pretest (females only) and pre-mating period and post-mating, animals were single housed in polycarbonate cages (Makrolon type III, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm), except for the exposure period. During the daily 6 hours of exposure, males returned to their home cage (Makrolon type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.
- Environmental Conditions:
Target temperatures: 23°C
Target humidity : 36 to 56%
Light: 12-hour light/12-hour dark cycle
- Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: Municipal tap water freely available to each animal via water bottles
- Veterinary Care: Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments were documented in the study records.

Administration / exposure

Route of administration:
dermal
Vehicle:
corn oil
Details on exposure:
The test item and vehicle were administered to the appropriate animals dermally once daily (for at least 6 hours, with a maximum of 7 hours) 7 days a week for a minimum of 28 days. Application was performed approximately the same time each day. The dose volume for each animal was based on the most recent body weight measurement. The test item formulation was applied onto a surgical gauze patch (Surgy 1D; less than or equal to 8 ply), with an area of approximately 10% of the total body surface (i.e. approx. 40 cm² for males and approx. 30 cm² for females). The gauze patch was mounted on Medical tape or Microfoam and held in contact with the skin with Tegaderm flexible bandage. To protect the application Lomir jackets were used, which were secured with Velcro (Manufacturers: Laboratoires Stella s.a., Liege, Belgium (surgical gauze) and 3M, St. Paul, Minnesota, U.S.A. (Medical Tape and Microfoam) and Lomir Biomedical, Hull, UK (Jackets)).
Details on mating procedure:
After 15 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. During the daily 6 hours of exposure, males returned to their home cage. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 postcoitum. Once mating had occurred, the males and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature protected from light for at least 30 minutes before application on the dressing.
All samples to be analyzed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility. Analyses were performed using a validated analytical procedure (Test Facility Study No.515251).
Duration of treatment / exposure:
- Males were treated for 29 days, up to and including the day before scheduled necropsy (included a minimum of 14 days prior to mating and during the mating period).
- Females that delivered were treated for 46-50 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy up to Day 19 post-coitum and starting on Day 4 of lactation (according to the OECD 422 guideline), up to and including the day before scheduled necropsy. The omission of exposure on Day 20 post-coitum up to PND 3 over a period of several weeks was not considered to affect the toxicological evaluation.
- Females which failed to deliver were treated for 37-38 days.
Frequency of treatment:
The test item and vehicle were administered to the appropriate animals dermally once daily (for at least 6 hours, with a maximum of 7 hours) 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control group)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females per group / 1 dose concentrations per group
Control animals:
yes, concurrent vehicle
Details on study design:
The dermal route of exposure was selected because this is the most likely route of human exposure during manufacture, handling or use of the test item.
The dose levels in this study were selected to be 0, 25, 75 and 250 mg/kg, based on the results of the dose range finder (Test Facility Study No. 515248, see Appendix 6). During the dose range finder animals dosed at 500 and 1000 mg/kg were sacrificed in extremis for animal welfare reasons. At 1000 mg/kg the treated skin became thicker and several skin lesions were noted and the males had also become hypersensitive to handling. The lesions of the skin were also considered an indication of a compromised integrity of the test system. During the second week of treatment, similar signs appeared in males at 500 mg/kg. Therefore the dose levels of 500 and 1000 mg/kg were considered unfeasible for dermal exposure.
Positive control:
none

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Any other clinical signs noted during the treatment period 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
effects observed, non-treatment-related
Description (incidence and severity):
Focal erythema and scales/scabs were noted at increased incidence on the treated skin in females and males at 250 mg/kg, respectively. As these clinical observations were mainly observed during the first weeks of treatment, they were regarded as non-adverse.

Procedure-related skin findings in the treated skin consisted of hyperplasia of the epidermis and the presence of pustule(s) and/or exudative inflammation. Since these microscopic findings were recorded at low degree in controls as well as in some high dose animals and can be seen as background findings in rats subjected to a repeated dose dermal study, they were regarded as procedure-related incidences (trauma to the wearing of the protective jacket).

Procedure-related skin findings were noted in untreated skin adjacent to the treated area (neck) and, less frequently, at the flank(s), back or shoulders. These findings were considered to be related to the treatment procedure (use of Tegaderm tape and protective jacket) and not test item-related as they occurred in all groups (including control) and without a dose-related pattern. Clinical findings included scales (also noted at necropsy), scabs, scars, wounds and focal/general erythema. Microscopic findings consisted of ulceration (minimal up to marked), hyperkeratosis (minimal), hyperplasia of the epidermis (minimal up to moderate), exudative inflammation (minimal up to marked) and intracellular edema (minimal) and/or the presence of a pustule (minimal). Since these findings were present across all groups (including controls), they were considered to be procedure-related (trauma to the wearing of the protective jacket).
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
One male at 75 mg/kg (no. 24) was sacrificed in extremis on Day 25 of treatment, as the exposure method was no longer tolerated. Main cause of moribundity was a (soring) wound on the shoulder (microscopic correlate: ulceration with crust formation) which was considered to be procedure-related (trauma to the wearing of the protective jacket).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain were considered not to be affected by treatment.
The statistically significant decrease in body weight gain observed in males at 25 mg/kg on Day 15 of treatment was partly attributed to one male (no. 16) that failed to gain weight over Days 8-15. This finding was considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption (before and after correction for body weight) was similar between treated and control animals over the treatment period.
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:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis:

A treatment-related statistical significant decrease in mean serum T4 level was noted in F0-males at 250 mg/kg (28% lower than concurrent control values). The mean value at 250 mg/kg level was below the historical control range. However, possible adversity of this effect could not be assessed within this type of screening study. Moreover, the available 28d oral study showing an adaptive response from the liver and its potential secondary effect on the thyroid following exposure to the substance would support a reversible and non adverse effect on the T4 levels observed.

Serum TSH levels showed a wide intragroup variation in all males and were increased unrelated to dose level when compared to the concurrent control group. At 25, 75 and 250 mg/kg, mean TSH levels of males were 3.4x, 1.7x and 3.1x higher than control values, respectively (statistically significant at 25 mg/kg). Means at 25 and 250 mg/kg were outside the range of the historical control data.
The tendency for higher serum TSH levels in treated males was not conclusive given the wide intragroup variation and the absence of a clear dose-related trend.

Serum levels of T4 and TSH in F0-females were considered unaffected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations.

Treated skin: There were minor microscopic findings in the treated skin consisting of hyperplasia of the epidermis, the presence of pustule(s) and/or exudative inflammation. These findings were noted at low degrees in control as well as some high dose animals and can be seen as a background findings in rats subjected to a repeated dose dermal study and were regarded procedure-related.

Procedure-related skin findings:
- Ulceration (minimal up to marked).
- Hyperkeratosis (minimal).
- Hyperplasia of the epidermis (minimal up to moderate).
- Exudative inflammation (minimal up to marked).
- Intracellular edema (minimal) and/or the presence of a pustule (minimal).
These lesions were mainly present in male rats and were recorded in controls as well as in test item-treated animals. Since these findings were present across all groups (including controls), they were considered to be procedure-related (trauma to the wearing of the protective jacket) and not test item-related. The ulceration with crust formation and/or ulcerative inflammation or pustule of the epidermis correlated to the scab formation recorded at necropsy.

All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain subjected to a dermal toxicity study.

Reproductive performance: Three couples had no offspring: 1/10 females at 25 mg/kg and 1/10 females at 250 mg/kg were not pregnant despite evidence of mating, and 1/10 females at 250 mg/kg had implantation sites only (she had a single implantation site). Histopathology did not reveal any abnormalities in the reproductive organs that could explain this reproductive failure.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was one finding of note: a nephroblastoma was recorded in male no. 19 (25 mg/kg), which correlated with the enlarged kidney with pelvic dilation observed at necropsy. As this neoplastic lesion can be seen as a background finding in rats of this age and strain and was only recorded in a single male of the low dose group, this finding was regarded to be unrelated to the treatment with the test item.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. One female of the 25 mg/kg group (no. 57) was acyclic. She had a normal precoital time and delivered a normal litter. Given its incidental nature and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was considered unaffected by treatment. All females showed evidence of mating (mating index 100%).

Details on results (P0)

Precoital Time: Precoital time was considered unaffected by treatment. All females showed evidence of mating within 4 days.
Number of Implantation Sites: Number of implantation sites was not affected by treatment. Low number of implantation sites were noted in one control female (no. 45: 2 implantation sites) and one female at 250 mg/kg (no. 75: one implantation site). Since all other pregnant females of the 250 mg/kg group presented with normal number of implantation sites and given the occurrence of this finding in the control group, this low number of implantation sites in one female at the highest dose level was regarded as unrelated to treatment.
Fertility Index: Fertility index was not affected by treatment. The fertility indices were 100, 90, 100 and 90% for the control, 25, 75 and 250 mg/kg group, respectively. Two females, one at 25 mg/kg (no. 60) and one at 250 mg/kg (no. 76) were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, they were considered incidental cases and, therefore, unrelated to treatment.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test item related and adverse effect observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Scabs, alopecia and (open) wounds were noted on different parts of the body throughout the lactation period in pups of one control litter (no. 49), 3 litters at 25 mg/kg (nos. 53, 56 and 59), 3 litters at 75 mg/kg (nos. 61, 64, 68) and 2 litters at 250 mg/kg (nos.77 and 79). Since they occurred in control and treated animals and showed no dose-related pattern, these findings in the pups were considered to be related to the wearing of Tegaderm tape and/or the jacket used for the dermal application by the dam rather than deficiencies in maternal care.
The nature and incidence of other clinical signs (including alopecia observed in litter nos. 48 (control) and 65 (Group 3)) remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant. Note: Only days on which clinical signs were present between first and last litter check are presented in the table.
Dermal irritation (if dermal study):
effects observed, non-treatment-related
Description (incidence and severity):
see clinical effects above.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Live birth index (number of live offspring on PND1 after littering as percentage of the total number of offspring born) was considered not to be affected by treatment. The live birth indices were 98% for the 25 mg/kg group and 100% for the other groups.
At first litter check, two pups at 25 mg/kg were found dead (both from litter no. 52). This pup mortality was considered to be unrelated to treatment as the mortality incidence did not show a dose-related trend and remained within normal limits.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered unaffected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At 250 mg/kg, serum Total T4 mean values of male and female pups were 21% and 10% lower than concurrent control values, respectively (statistically significant in males only). As mean values remained within the historical control data4, the observed decrease in T4 values at this high dose level was considered of non-toxicological relevance.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio was considered unaffected by treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered unaffected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 250 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Macroscopic findings such as scabs and wounds were noted in pups of two litters at 25 mg/kg (nos. 53 and 59), three litters at 75 mg/kg (nos. 61, 64 and 68) and one litter at 250 mg/kg (no. 77). These gross findings in the pups were considered to be related to the wearing of Tegaderm tape and/or the jacket used for the dermal application by the dam rather than deficiencies in maternal care.
The nature and incidence of other macroscopic findings remained within the range considered normal for pups of this age and were, therefore, not considered to be related to treatment.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test item related and adverse effect observed

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Nympheal were established:
- Parental , Reproduction and Developmental NOAEL: At least 250 mg/kg
A dose level of 500 mg/kg or higher could not be tested as the animals would not tolerate this dose level and the integrity of the dermal exposure would be compromised (based on the dose range finder).
Executive summary:

The objectives of this study were to determine the potential toxic effects of Nympheal when given dermally for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. The dose levels in this study were selected to be 0, 25, 75 and 250 mg/kg, based on the results of the dose range finder (Test Facility Study No. 515248). During the dose range finder animals dosed at 500 and 1000 mg/kg were sacrificed in extremis for animal welfare reasons. At 1000 mg/kg the treated skin became thicker and several skin lesions were noted and the males had also become hypersensitive to handling. The lesions of the skin were also considered an indication of a compromised integrity of the test system. During the second week of treatment, similar signs appeared in males at 500 mg/kg. Therefore the dose levels of 500 and 1000 mg/kg were considered unfeasible for dermal exposure.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight (gain) and food consumption, estrous cycle length and regularity, serum level of thyroid hormones T4 and TSH (F0-males and F0-females), gross necropsy findings, organ weights (thyroid and male reproductive organs) and histopathologic examinations (skin, thyroid and reproductive organs).

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, serum levels of thyroid hormoneT4 (PND 14-16 pups)). Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

Parental results

No parental toxicity was observed up to the highest dose level tested (250 mg/kg). Focal erythema and scales/scabs were noted at increased incidence on the treated skin in females and males at 250 mg/kg, respectively. As these clinical observations were mainly observed during the first weeks of treatment, they were regarded as non-adverse.

A test item-related decrease in serum T4 levels was noted in males at 250 mg/kg (28% lower than control values). The tendency for higher serum TSH levels in treated males was not conclusive given the wide intragroup variation and the absence of a clear dose-related trend. Males showed no changes in thyroid weight or morphology. Serum levels of T4 and TSH in females were not considered to be affected by treatment.

Procedure-related skin findings in the treated skin consisted of hyperplasia of the epidermis and the presence of pustule(s) and/or exudative inflammation. Since these microscopic findings were recorded at low degree in controls as well as in some high dose animals and can be seen as background findings in rats subjected to a repeated dose dermal study, they were regarded as procedure-related incidences (trauma to the wearing of the protective jacket).

Procedure-related skin findings were noted in untreated skin adjacent to the treated area (neck) and, less frequently, at the flank(s), back or shoulders. These findings were considered to be related to the treatment procedure (use of Tegaderm tape and protective jacket) and not test item-related as they occurred in all groups (including control) and without a dose-related pattern. Clinical findings included scales (also noted at necropsy), scabs, scars, wounds and focal/general erythema. Microscopic findings consisted of ulceration (minimal up to marked), hyperkeratosis (minimal), hyperplasia of the epidermis (minimal up to moderate), exudative inflammation (minimal up to marked) and intracellular edema (minimal) and/or the presence of a pustule (minimal). Since these findings were present across all groups (including controls), they were considered to be procedure-related (trauma to the wearing of the protective jacket).

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (250 mg/kg).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (250 mg/kg).

Based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Nympheal were established:

- Parental , Reproduction and Developmental NOAEL: At least 250 mg/kg  (Note: Serum levels of total T4 were decreased at 250 mg/kg (statistically significant for males only) which was regarded as test item-related. However, possible adversity of this effect could not be assessed within this type of screening study and was, therefore, not taken into account when determining the parental NOAEL).

A dose level of 500 mg/kg or higher could not be tested as the animals would not tolerate this dose level and the integrity of the dermal exposure would be compromised (based on the dose range finder).