Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August - 24 November 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD Guideline 414 with deviations: tested at two dose-levels only; no data on rationale for vehicle selection
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
tested at two dose-levels only; no data on rationale for vehicle selection
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Mexoryl SAB
- Physical state: Fine white powder
- Analytical purity: see confidential details
- Lot/batch No.: Op T 4E
- Date of receipt: 18 April 1995
- Storage condition of test material: Stored at room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK. Ltd., Margate, England
- Age at study initiation: Approximately 8-10 weeks
- Weight at study initiation: 201-225 g
- Housing: The males (before mating) and females (after mating and during the dosing period) were individually housed in grid-bottomed polypropylene cages suspended over paper-lined trays. During acclimatisation and until pairing for mating, the females were group housed, in fives, in stainless steel cages with grid-bottoms suspended over paper-lined trays. From day 16 of pregnancy, the mated females were housed in solid-bottomed cages with graded softwood sawdust provided as bedding.
- Diet (e.g. ad libitum): Pelleted diet SQC Rat and Mouse No. 3 Breeder (expanded) (Special Diets Services Limited, Witham, Essex, U.K.), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-22 °C
- Humidity: 46-61 %
- Air changes: 16 air changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 25 August 1995 To: 25 September 1995

Administration / exposure

Route of administration:
dermal
Vehicle:
other: PEG 300
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area
- % coverage: Not less than 10 % of the body surface area
- Type of wrap if used: No wrap used; formulations were applied directly to a shaved area of skin on the back.
- Time intervals for shavings: Approximately 24 h before the start of dosing, fur was removed from the dorsal area by shaving. During the study period, the application sites were shaved as necessary to maintain them free of fur.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Test site was washed, where practicable, using warm water to remove residual test article and the skin was blotted dry with a paper towel.
- Time after start of exposure: After approximately 6 h of exposure

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw
- Constant volume or concentration used: No, dose volume given to each animal was adjusted daily according to bodyweight.

PREPARATION OF DOSING SOLUTIONS
- Fresh formulations were prepared daily by suspending the weighed amount of test article in the appropriate weight of vehicle.

VEHICLE
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes; In order to prevent ingestion of the test article, Elizabethan collars were fitted daily, from immediately after application of the dose and were left in place for the exposure period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Sampling and analytical method: Duplicate samples were taken of each formulation prepared on the first day of dosing and on one day towards the end of the dosing period, and analysed using a transferred HPLC method with UV detection. In addition, two samples (1mL) were taken from each formulation prepared (including that of the control group) and stored frozen (at less than -18 °C) until production of the final report.
- Results: The test article formulations were chemically stable for 24 h when stored under ambient conditions in the dark at concentrations of 20 and 50 mg/mL. Analysed concentrations were within 9 % of the nominal concentrations. Test article formulations were therefore considered to have been accurately prepared.
Details on mating procedure:
- Impregnation procedure: Cohoused
- F/M ratio per cage: 2 F/1 M
- Females were mated within four consecutive days.
- Proof of pregnancy: Sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Days 6-15 of gestation
Frequency of treatment:
Once daily
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40 and 100 mg/kg bw/day (equivalent to 0, 2 and 5 % Mexoryl SAB)
Basis:
other: topically applied daily
No. of animals per sex per dose:
24 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the existing toxicity data and on the basis of results of a preliminary study conducted at the laboratory (Study no. LRL/101/C).
- Rationale for animal assignment (if not random): Animals were allocated to dose groups using a randomisation procedure based on bodyweight.

Examinations

Maternal examinations:
MORTALITY: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity.

CLINICAL OBSERVATIONS: Yes
- Time schedule: All visible signs of reaction to treatment were recorded daily from Day 0 of pregnancy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6 to 15 inclusive and 20 of pregnancy

FOOD CONSUMPTION: Yes
- Food consumption was measured over the following periods: Days 0-6, 6-9, 9-12, 12-15 and 15-20 of pregnancy.

POST-MORTEM EXAMINATIONS: Yes
- The females were sacrificed on gestation Day 20 by CO2 asphyxiation and a necropsy was performed.
- Organs or tissues showing macroscopic abnormalities were removed and fixed in neutral buffered formaldehyde. The treated skin and a section of control skin (taken from the right hind limb) from all animals were fixed and stored in neutral buffered formaldehyde. These tissues were not examined microscopically.

OTHER:
- Assessment of skin reaction: Appearance of the application sites were assessed daily from Day 6 to 20 of pregnancy and skin reactions were scored according to the same scale as recommended by OECD Guideline 404.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea
- Number and distribution of implantation sites: The implantations were classified as early resorptions, late resorptions, dead foetuses or live foetuses and were numbered separately for the right (R) and left (L) horns.
- Foetal weights and sexes (live foetuses)
Fetal examinations:
- External examinations: Yes [all living foetuses]; weighed and examined for externally visible malformations
- Visceral and skeletal examinations: Yes [half per litter]; fixed in 70% alcohol, dissected and examined for visceral abnormalities. Foetuses were then eviscerated and the carcasses cleared with KOH, stained with Alizarin Red S and examined for skeletal abnormalities.
- Soft tissue examinations: Yes [all the remaining foetuses]; fixed in Bouin's fluid and the isolated head, heart, kidneys and all major organs and blood vessels were microdissected.
Statistics:
- Bodyweights and bodyweight gains, food consumption, numbers of corpora lutea, implantation sites and live foetuses and foetal bodyweight (by sex and on litter basis): Analysed by ANOVA (Analysis of variance) and Williams' test
- Pre-implantation loss, post-implantation loss and foetal sex ratios (on litter basis): Analysed by Kruskal-Wallis and Shirley's test
- Foetal abnormalities (on litter basis): Analysed by Kruskal-Wallis and Fisher's Exact test
Indices:
- Pre-implantation loss (%) = (total number of corpora lutea - number of implantation sites) / total number of corpora lutea X 100
- Post-implantation loss (%) = (number of implantation sites - number of live foetuses) / number of implantation sites X 100
Historical control data:
- Background incidence of foetal abnormalities in historical developmental toxicity studies performed in the laboratory (July 1990 – September 1993) on the Sprague-Dawley rats were given as reference.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortalities and clinical observations: No death was noted and clinical signs were limited to red perinasal and periorbital fur staining during the dosing period in all groups (including control).
- Body weight: No treatment-related effect was noted on mean maternal body weight. However, compared to controls, a statistically significant reduction in body weight gain values was observed at both doses (11.8% at 40 mg/g bw/d and 23.5% at 100 mg/kg bw/d) over Days 9-12 only, and Days 6-15 of pregnancy in the highest dose group (due to the body weight reduction over GD9 to GD11 only).
- Food consumption: No significant treatment-related changes were noted.
- Skin reactions: At 100 mg/kg bw/day, slight to severe erythema (score 3 from the 6th day of administration) with slight eschar formation and very slight oedema were noted at the dosing site during the dosing and post-dosing periods and it persisted until Day 20 of pregnancy in majority of females.
At 40 mg/kg bw/day, slight to moderate erythema with eschar formation and/or desquamation were noted at the dosing site during the dosing and post-dosing periods and it persisted until Day18 of pregnancy in majority of females.
These abnormalities occurred with dose-related severity.
- Necropsy: At necropsy, treatment-related scabbing and/or reddening were noted at the dosing site for all females in 100 mg/kg bw/day group and scabbing was noted at the dosing site in 8 females in 40 mg/kg bw/day group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
local effect
Effect level:
< 40 mg/kg bw/day
Based on:
test mat.
Remarks:
topically applied
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Remarks:
systemic effect
Effect level:
> 100 mg/kg bw/day
Based on:
test mat.
Remarks:
topically applied
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
> 100 mg/kg bw/day
Based on:
test mat.
Remarks:
topically applied
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
PREGNANCY DATA:
- No significant treatment-related changes were noted in mean numbers of corpora lutea, implantations, live foetuses, mean foetal sex ratio and the extent of pre- and post-implantation losses.
- At 40 mg/kg bw/day, a slightly higher post-implantation was noted but it was considered incidental and unrelated to treatment.

FOETAL WEIGHTS:
- No significant treatment-related changes were noted.
- Compared to control, a non-significantly slightly lower mean weight was noted at 40 mg/kg bw/day but it was considered incidental and unrelated to treatment.

FOETAL EXAMINATION:
- Major abnormalities: No significant treatment-related changes were noted. Few incidences of abnormalities (1, 5 and 0 at 0, 40 and 100 mg/kg bw/day, respectively) included heart and major blood vessel abnormalities, lung abnormalities, situs inversus and major fusion of the ribs.
- External and visceral minor abnormalities: No significant treatment-related changes were noted.
- Skeletal minor abnormalities: No significant treatment-related changes were noted. Compared to background range, a higher incidence of skeletal minor abnormalities and variants (i.e. degree of ossification of the hyoid, thoracic centra, sacral neural arch, caudal centra, stemebrae, pelvic girdle, metatarsals and metacarpals) were observed in all groups including control (incidence of 18.3% in controls, 24.3% at 40 mg/kg bw/d and 31.7% at 100 mg/kg bw/day).
Compared to background range, a higher but not statistically significant increase of incidence of skeletal minor abnormalities and variants including incomplete ossification of the sacral neural arch was observed in all groups including control. Indeed, the historical control range was from 0 to 9% while the incidence of sacral neural arch incomplete ossification in the vehicle controls of the study was 18.3%. Therefore, it’s likely that the incidence of the skeletal minor abnormalities observed in both treated groups were also overestimated. Moreover, in the absence of other developmental effects, the overestimated and statistically non-significant skeletal findings observed in both treated groups were likely related to the transient decrease in bodyweight gain of the dams (GD9 to GD12) attributed itself to the moderate or severe skin lesions leading to pain and stress in animals.

- For more details, refer tables 8, 9 and 10 of the attached PDF document titled 'Figures and tables'

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 7.8.2/1: Group mean maternal bodyweight gains (g) ± S.D.

Dosage
(in mg/kg bw/day)

N

Days of pregnancy

0-6

6-7

6-8

6-9

9-12

12-15

15-20

6-15

0 (control)

23

27 ± 8

1 ± 2

5 ± 2

8 ± 3

17 ± 4

19 ± 3

67 ± 9

44 ± 7

40

22

27 ± 7

2 ± 3

5 ± 5

9 ± 5

15 ± 4*

17 ± 4

62 ± 11

40 ± 10

100

24

25 ± 6

2 ± 3

5 ± 5

8 ± 5

13 ± 3***

17 ± 3

62 ± 8

38 ± 8*

N = number of animals included in the mean

* = statistically significantly different from control, p < 0.05, Williams' test

*** = statistically significantly different from control, p < 0.001, Williams' test

Applicant's summary and conclusion

Conclusions:
Under the test conditions, neither systemic effect as body weight gain reduction in females nor developmental effect in offspring as higher skeletal minor abnormalities and variants incidence could be attributed to the test substance itself. However, these effects could probably be related to the severe skin lesions leading to pain and stress in animals.
This was supported by the results observed in the preliminary study performed in rats exposed by dermal route at the same doses for 10 days (see % 7.5.3). Indeed, no body weight gain reduction was measured in animals while minimal irritation was observed (characterised by slight to well-defined erythema and edema) in the highest dose group (i.e. 5% or 100 mg/kg bw/day).
Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414, groups of pregnant female Sprague-Dawley rats (24/dose) were dosed once daily, by dermal application, with Mexoryl SAB at dose levels of 0 (vehicle), 40 and 100 mg/kg bw/day in PEG 300 from Days 6 to 15 of pregnancy. The formulations were applied directly to the shaved area of skin on the back (not less than 10 % of the body area). After approximately 6 h, the test site was washed using warm water to remove residual test article and the skin was then blotted dry with a paper towel. All visible signs of reaction to treatment and the appearance of the application sites were assessed and recorded daily. Maternal clinical signs, bodyweights and food consumption were recorded. On Day 20 of pregnancy, all females were killed for necropsy and a skin section was removed and fixed in neutral buffered formaldehyde. Numbers of corpora lutea and live and dead implantations were recorded. Live foetuses were weighed, sexed and examined for external and visceral abnormalities. One half of the foetuses were subsequently examined for skeletal abnormalities.

No premature deaths or treatment-related clinical signs were recorded. Animals from the treated groups only were observed with erythema and eschar formation that occurred with dose-related severity and slight oedema during the dosing and post-dosing periods. No treatment-related effect was noted on mean maternal food consumption and body weight. However, compared to control, a statistically significant reduction in body weight gain values were observed at 40 and 100 mg/kg bw/day over Days 9-12 and Days 6-15 of pregnancy at the highest dose. At necropsy, treatment-related scabbing and/or reddening were noted at the dosing site in both groups. No treatment-related effects were noted on pregnancy parameters, mean foetal weight (male, female and total) and incidences of major external, visceral or skeletal abnormalities. Compared to background range, a higher but not statistically significant increase of incidence of skeletal minor abnormalities and variants including incomplete ossification of the sacral neural arch was observed in all groups including control. Indeed, the historical control range was from 0 to 9% while the incidence of sacral neural arch incomplete ossification in the vehicle control group was 18.3%. Therefore, it’s likely that the incidence of the skeletal minor abnormalities observed in both treated groups were also overestimated. Moreover, theses statistically non-significant skeletal findings observed in both treated groups were likely related to the transient but statistically significant decrease in bodyweight gain of the dams (GD9 to GD12) attributed itself to the moderate or severe skin lesions leading to pain and stress in animals. Therefore, the increase in the incidence of skeletal variations reported in foetuses at both doses were likely secondary to maternal toxicity (induced by local effects leading to pain and stress) and not indicative of a teratogenic effect.

This statement was supported by the results observed in the preliminary study performed in rats exposed by dermal route at the same doses for 10 days (see % 7.5.3). Indeed, no body weight gain reduction was measured in animals while minimal irritation was observed (characterised by slight to well-defined erythema and edema) in the highest dose group (i.e. 5% or 100 mg/kg bw/day).

Under the test conditions, maternal toxicity characterised by severe skin lesions at the site of administration was noted in female Sprague-Dawley rats topically applied with Mexoryl SAB at dose levels of 40 and 100 mg/bw/day. Neither systemic effect as body weight gain reduction in the females nor effect in foetuses as higher skeletal minor abnormalities and variants incidence could be attributed to the test substance itself. However, these effects could probably be related to the severe skin lesions leading to pain and stress in the dams.