2-hydroxy-5-octanoylbenzoic acid

Endpoint summary

Description of key information

In a preliminary 10-day dermal toxicity study conducted similarly to the OECD Guideline 410 and in compliance with GLP , NOEL for local effect was 30 mg/kg bw/d (2%) and NOEL for systemic effect was higher to 100 mg/kg bw/d (5%). Considering a rat body weight of 0.25 kg and an exposure surface of 10% (i.e. 45 cm2), a NOEL of 30 mg/kg bw/d corresponded to 167 mg/cm2.
In a 4 week oral toxicity study performed in rats in accordance with OECD guideline 407 and in compliance with GLP, NOEL for local effect was 30 mg/kg bw/d and NOEL for systemic effect was higher to 100 mg/kg bw/d considered as the highest dose bearable for the animals without suffering.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 1993 to 09 August 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study following OECD guideline 407. However, dose level was reduced from 300 mg/kg bw/day to 200 mg/kg bw/day from day 13 of the treatment (death occured). Temperature also dropped to 18°C on two occasions and several organs were not weighed (thymus, spleen, brain and heart)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Dose level reduced from 300 mg/kg/day to 200 mg/kg/day from day 13 of the treatment, temperature dropped below 19°C at two occasions, several organs not weighed (thymus, spleen, brain and heart)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CRL:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River limited, Margate, U.K.
- Age at study initiation: 3-4 weeks
- Weight at study initiation: 124-160 g for males, 112-148 g for females
- Housing: in groups of 5 in stainless steel cages
- Diet (e.g. ad libitum): SQC Rat and Mouse maintenance Diet No. 1, Expanded (Special Diets Services Limited, Witham, England), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23
- Humidity (%): 40-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 June 1993 To: 09 August 1993

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: separate preparations were prepared freshly each day, for each dose level

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 1, 3, 10 and 30 (then 20) mg/mL for 10, 30, 100 and 300 (then 200) mg/kg bw/d groups
- Amount of vehicle (if gavage): 10 mL/kg bw
- Fluka Chemicals Ltd, Gillingham, U.K.; Batch numbers 322054/1393 and 322054/11292

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the preparations were checked. Concentrations were measured by HPLC using a detection wavelength of 274 nm.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 then 200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 for control and the highest dose level
5 for the 3 other dose levels (10, 30 and 100 mg/kg bw/d)

Control animals:

yes

Details on study design:

- Dose selection rationale: based on the existing toxicity data
- Due to deaths and adverse clinical signs dose level was reduced from 300 to 200 mg/kg bw/d from day 13

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day including weekends
- Cage side observations were checked for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: animals weighed at the start of the study and then weekly and at necropsy (see table 1)

FOOD CONSUMPTION
- Food consumption for each cage was determined weekly and mean diet consumption was calculated as g food/animal/week (see table 2)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: only once, 2 h after dosing, during week 4 of treatment
- Anaesthetic used for blood collection: no data
- Animals fasted: overnight
- How many animals: all surviving animals
- Parameters checked: haemoglobin concentration, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total leucocyte count, platelet count, leucocyte differential count, reticulocyte count (smear prepared but not read)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: only once, 2 h after dosing, during week 4 of treatment
- Animals fasted: overnight
- How many animals: all surviving animals
- Parameters checked were blood urea nitrogen, glucose, alkaline phosphatase, alanine amino transferase, aspartate amino transferase, total protein, albumin, albumin/globulin ratio, sodium, potassium, calcium, chloride, inorganic phosphorous, cholesterol, triglycerides, total bilirubin, gamma glutamyl transferase and creatinine.

Sacrifice and pathology:

GROSS PATHOLOGY: all animals were weighed and examined externally. A macroscopic examination was then performed by opening the cranial, toracic and visceral cavities and by observing tissues in situ. The following organs were weighed for all animals: adrenals, kidneys, liver and testes.
HISTOPATHOLOGY: gross lesions as well as adrenals, heart, kidneys, liver and spleen in the control and the highest dose groups were examined microscopically. Because of histopathological lesions, stomach and gross lesions were examined for all animals.

Other examinations:

no data

Statistics:

Bodyweight gain, haematological and organ weight data were evaluated by analysis of variance and, if a between groups difference significant at the 5% level occurred, by pairwise t-tests between the control and treatment groups.
Biochemistry and haematology data were tested using the Kruskal-Wallis one way analysis of variance by ranks test at the 5% (0.05) significance level. If a significant Kruskal-Wallis value was obtained, the control group was compared with the test groups using multiple comparison techniques, to determine if the significant differences was between the control group and one or more of the test groups.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were 5 deaths at the highest dose (3 males at 300 mg/kg bw/d, 1 male and 1 female at 200 mg/kg bw/d). Clinical signs prior to death were noisy and irregular/fast respiration.
Rough coat, piloerection and post dose salivation observed in all animals of the highest dose group. Rough coat and piloerection was also noted in the 100mg/kg/day group.

BODY WEIGHT AND WEIGHT GAIN (See table 1)
A slight (13-14%) reduction in bodyweight gain over the treatment period as a whole, was noted for males from the highest dose group and females from 100 mg/kg/d group. Other treated groups had bodyweight gains similar to controls throughout the treatment period. Bodyweight gain for males from the highest dose group was also reduced during the treatment-free period, when compared to controls (16%). Females from the highest dose group had bodyweight gains slightly superior to controls during the treatment-free period.

FOOD CONSUMPTION (See table 2)
Food consumption was reduced slightly for males from the highest dose group. This was concomittant with the reduction in bodyweight observed for this group. The reduction was apparent in both the treatment and treatment-free period. All other treated groups had food consumption values similar to controls throughout the treatment period.

HAEMATOLOGY
No significant changes were observed in haematology.

CLINICAL CHEMISTRY
Statistically significant changes observed in blood chemical parameters were within the normal range found in the laboratory and were considered to be unrelated to test article administration.

ORGAN WEIGHTS
Absolute and bodyweight-related organ weights were unaffected.

GROSS PATHOLOGY
A dose-related increase in abnormalities of the stomach (abnormal shape, colour and consistency) was noted for animals of both sexes from 100 and 300/200 mg/kg bw/d groups. This was still present in some animals following the treatment-free period.

HISTOPATHOLOGY: NON-NEOPLASTIC (See table 3)
Hyperplasia of the non-glandular stomach was observed for both sexes from the highest dose group; this was accompanied by chronic inflammation and ulceration. Similar but less severe hyperplastic lesions were observed in one male from the 100 mg/kg bw/d group and all recovery animals of the highest dose group.

Dose descriptor:

NOEL

Remarks:

for local effect

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Dose descriptor:

NOEL

Remarks:

for systemic effect

Effect level:

> 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effect observed in males and females up to the highest dose bearable for the animals, i.e. without mortality or without severe gastro-intestinal tract irritation.

Critical effects observed:

not specified

Table 1: Bodyweights and body weight gains (g) - group mean values

Group and sex	Week number						Gain	Week 5			Gain
		1	2	3	4	5	1-5	recovery	6	7	5-7
Control Males	Mean S.D.	142 8.8	193 14.9	242 22.7	263 22.6	309 30.2	167 27.5	307 44.4	338 6.8	374 48.6	67 12.2
10 mg/kg bw/d Males	Mean S.D.	145 5.9	181 27.8	233 31.4	254 34.5	300 36.1	155 34.5		- -	- -	- -
30 mg/kg bw/d Males	Mean S.D.	145 10.3	198 17.9	247 26.8	268 29.8	317 35.2	172 25.5		- -	- -	- -
100 mg/kg bw/d Males	Mean S.D.	139 9.4	182 14.5	229 18.7	253 28.8	298 37.0	160 32.6		- -	- -	- -
300/200 mg/kg bw/d Males	Mean S.D.	136 7.0	179 17.5	226 18.5	245 24.4	281 21.4	146 17.8	267 6.1	288 7.2	323 7.6	56 5.5
Control Females	Mean S.D.	131 10.5	165 7.4	191 10.0	199 12.7	232 14.5	101 13.4	232 8.8	242 10.2	266 11.1	33 7.9
10 mg/kg bw/d Females	Mean S.D.	133 9.3	165 13.3	192 12.1	197 11.8	231 17.5	99 11.1		- -	- -	- -
30 mg/kg bw/d Females	Mean S.D.	129 13.0	160 9.8	185 12.0	193 10.0	230 14.0	101 6.5		- -	- -	- -
100 mg/kg bw/d Females	Mean S.D.	127 16.2	154 12.3	178 14.8	188 11.5	214 11.0	87 8.2		- -	- -	- -
300/200 mg/kg bw/d Females	Mean S.D.	128 8.6	156 13.0	182 18.4	199 15.0	225 18.3	96 16.4	223 16.0	234 20.1	261 20.0	38 4.7

Table 2: Food consumption (g/animal /week) - group mean values

Group and sex	Week number	1	2	3	4	Total food eaten Weeks 1-4	% of control	5	6	Total food eaten weeks 5-6	% of control
Control males	Mean S.D.	169 2.1	191 3.5	179 2.1	182 2.8	721 -	- -	218 -	216 -	434 -	- -
10 mg/kg bw/d males	Mean	157	196	187	185	725	101	-	-	-	-
30 mg/kg bw/d males	Mean	170	196	177	182	725	101	-	-	-	-
100 mg/kg bw/d males	Mean	150	184	170	182	686	95	-	-	-	-
300/200 mg/kg bw/d males	Mean S.D.	153 14.8	160 19.1	166 10.6	171 7.1	650 -	90 -	196 -	194 -	390 -	90 -
Control females	Mean S.D.	139 6.4	149 7.1	137 7.1	149 7.8	574 -	- -	158 -	157 -	315 -	- -
10 mg/kg bw/d females	Mean	142	153	136	144	575	100	-	-	-	-
30 mg/kg bw/d females	Mean	135	141	128	145	549	96	-	-	-	-
100 mg/kg bw/d females	Mean	134	141	126	139	540	94	-	-	-	-
300/200 mg/kg bw/d females	Mean S.D.	137 5.7	147 3.5	131 7.1	142 2.8	557 -	97 -	176 -	169 -	345 -	110 -

Table 3: Microscopic pathology

Group	Control males	Control recovery males	10 mg/kg bw/d males	30 mg/kg bw/d males	100 mg/kg bw/d males	300/200 mg/kg bw/d males	300/200 mg/kg bw/d recovery males		Control females	Control recovery females	10 mg/kg bw/d females	30 mg/kg bw/d females	100 mg/kg bw/d females	300/200 mg/kg bw/d females	300/200 mg/kg bw/d recovery females
No. examined	5	5	5	5	5	7	3		5	5	5	5	5	5	5
Squamous epithelial hyperplasia
Slight	0	0	0	0	1	1	2		0	0	0	0	0	0	5
moderate	0	0	0	0	0	3	1		0	0	0	0	0	1	0
marked	0	0	0	0	0	2	0		0	0	0	0	0	4	0
Chronic inflammation	0	0	0	0	0	3	0		0	0	0	0	0	4	0
Ulceration	0	0	0	0	0	2	0		0	0	0	0	0	1	0

Conclusions:

Under the test conditions of this study, severe local effects were observed in the non-glandular stomach of rats administered 300/200 mg/kg bw/d and at 100 mg/kg bw/d with less severity. At lower doses (10 and 30 mg/kg bw/d), no local effects were observed in any animals. No systemic effect was observed at any doses up to the highest dose bearable for the animals corresponding to 100 mg/kg bw/d. Hence, NOEL for local effect was 30 mg/kg bw/d while NOEL for systemic effect was higher than 100 mg/kg bw/d considered as the highest dose exposure that it was ethically acceptable to use.

Executive summary:

In a 4 week oral toxicity study performed in accordance with OECD guideline n° 407 and in compliance with GLP, Mexoryl SAB was administered through gavage to groups of 5 Crl:CD (SD) rats/sex/dose mixed in polyethylene glycol (PEG 300) at dose levels of 0, 10, 30, 100 and 300 (then 200) mg/kg bw/day for 4 weeks. 2 recovery groups of 5 animals/sex/group (for control and the highest dose groups) were maintained undosed for further 14 days after the 4-week treatment. The dose level of the high dose group was dropped from 300 to 200 mg/kg bw/day on day 13 of the treatment period because of adverse clinical signs, including death.

Animals were observed daily, bodyweights and food consumption were recorded weekly. Blood samples were obtained from all surviving animals during week 4. At termination all animals were subjected to a detailed necropsy during which the weights of several organs were recorded and a wide range of tissues was preserved. A microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. Gross lesions were examined from all animals killed at the end of the treatment period, together with stomachs from all dose groups including the recovery animals.

There were 5 deaths in the highest dose group during treatment. Rough coat, piloerection and post dose salivation were noted in the highest dose group. Bodyweight gain was reduced slightly (13-14 %) for males of the highest dose group during the treatment and treatment-free periods and for females given 100 mg/kg bw/day during the treatment period only. Males given the highest dose had reduced food consumption during both the treatment and treatment-free period. Haematology and blood chemistry examinations did not reveal any effects. Macroscopic observations at necropsy revealed a dose-related increased incidence of stomach abnormalities for animals given 100 and 300/200 mg/kg bw/day at the end of both the treatment and treatment-free period. Microscopic pathology revealed hyperplasia of the non-glandular stomach for animals given 300/200 mg/kg bw/day, accompanied by chronic inflammation and ulceration. One male given 100 mg/kg bw/day also showed hyperplasia. Similar but less severe hyperplastic lesions were also observed for the recovery animals previously given 300/200mg/kg bw/day. The hyperplasia of the non glandular mucosa of the stomach was less severe at the end of the treatment-free period indicating that some recovery had taken place. Also, reversibility was shown and the effects are limited to the non-glandular stomach, therefore they are of doubtful relevance to humans.

Hence, the results showed severe local effects in the non-glandular stomach of animals administered 300/200 mg/kg bw/d and at 100 mg/kg bw/d with less severity (one male). At lower doses (10 and 30 mg/kg bw/d), no local effects were observed. No systemic effect was observed at any doses up to the highest dose bearable for the animals corresponding to 100 mg/kg bw/d (no severe non-glandular stomach irritation leading to death). Hence, NOEL for local effect was 30 mg/kg bw/d while NOEL for systemic effect was higher to 100 mg/kg bw/d considered as the highest dose bearable for the animals.

Under the test conditions of this study, NOEL for local effect was 30 mg/kg bw/d and the NOEL for systemic effect was higher than 100 mg/kg bw/d. This dose was considered as the highest exposure that it was ethically acceptable to use.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information as a whole meets the tonnage driven data requirement of REACH. Moreover, reliability and consistency are observed across the different studies.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 June – 28 July, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Preliminary study performed in GLP laboratory similarly to OECD Guideline 410, used as a range-finder experiment for an OECD Guideline 414 study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

tested at 2 dose levels only; duration of exposure: 10 days; only female animals were used; haematological tests, clinical biochemistry tests and detailed histopathological examinations were not followed

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)BR (VAF plus)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK. Ltd., Margate, England
- Weight at study initiation: 200-300 g
- Housing: Housed individually in grid-bottomed polypropylene cages
- Diet (e.g. ad libitum): Pelleted diet SQC Rat and Mouse No. 1 (expanded) (Special Diets Services Limited, Witham, Essex, U.K.), ad libitum
- Water (e.g. ad libitum): Tap water (in bottles), drawn directly from the water mains, ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 40-69 %
- Air changes: 16 air changes/h
- Photoperiod: 12 h dark/ 12 h fluorescent light

Type of coverage:

open

Vehicle:

other: PEG 300

Details on exposure:

TEST SITE
- Area of exposure: Clipped area of the back
- % coverage: Not less than 10 % of the body area
- Type of wrap if used: Test article formulations were applied directly to the skin of the clipped area of the back.
- Time intervals for shavings or clipplings: Approximately 24 h before the start of dosing, fur (equivalent to not less than 10 % of the surface area of the body) was removed from the dorsal area by clipping or shaving. During the dosing period, the application sites were clipped as often as was necessary to maintain them free of fur.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Test sites were washed using warm water to remove residual test article and the skin was then blotted dry with a paper towel.
- Time after start of exposure: Approximately 6 h

TEST MATERIAL
- Constant volume or concentration used: Yes, 2 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, animals were fitted with Elizabethan collars for the 6 h exposure period in order to prevent ingestion of the test article.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

6 h/day; 10 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 2 and 5 %
Basis:
other: directly applied on skin

No. of animals per sex per dose:

Five virgin females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Selected females were allocated randomly to treatment group using a stratified bodyweight procedure.

Positive control:

No

Observations and examinations performed and frequency:

MORTALITIES:
- Time schedule: Twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded over Days 1-4, 4-7 and 7-10 of the study

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Daily

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all surviving animals were killed by CO2 asphyxiation at the end of treatment period and were submitted to the procedure of necropsy including the following examinations: appearance of tissues in situ from the cranial, thoracic and abdominal cavities.
HISTOPATHOLOGY: Treated skin and a section of control skin (taken from the right hind limb) from all animals was fixed and stored in neutral buffered formaldehyde. These tissues were not examined microscopically.

Other examinations:

None

Statistics:

Analysis of variance (ANOVA) was performed on all parameters, using treatment group as the factor in the analysis. Residuals from this preliminary analysis were examined for heterogeneity of variance using Levenes test. If Levenes test was significant at the 1 % level, then the particular variable concerned was subjected to a non-parametric analysis. Otherwise, William’s test was performed to compare the high dose with control at the two-sided 5 % level. If this test was statistically significant then comparisons of the subsequent doses against control was performed at the one-sided 5 % level until a non-significant difference was found, at which point the testing stopped. The highest dose at which the difference from control was non-significant was deemed to be the no-effect level.
If Levenes test indicated that there were significant differences in the treatment group variances or if a parametric analysis was deemed to be inappropriate, then a Kruskal-Wallis ANOVA was performed to assess overall differences between the treatment groups, followed by Shirley's nonparametric version of William’s test, which is based on mean ranks rather than the arithmetic means.

Clinical signs:

effects observed, treatment-related

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
- No premature death was noted.
- Animals from the group treated at 5 % were observed to be scratching at the application site from Day 8 of the dosing period onwards.
- No other treatment-related clinical observation was noted.

DERMAL IRRITATION:
- Between 2 and 5 animals from the group treated at 5 % were observed with very slight erythema from Day 6 onwards and one female was observed with well-defined erythema between Days 8 and 10 of the dosing period. In addition, between 3 and 4 animals from this group were observed with very slight oedema from Day 7 of the dosing period. There were no skin reactions in either of the other groups.

BODY WEIGHT AND WEIGHT GAIN:
- No effect of treatment was noted on bodyweight gain throughout the dosing period.
- Occasionally, transient weight losses were observed which, because of the small group sizes, skewed the means.

FOOD CONSUMPTION:
- No effect of treatment was noted on mean food consumption.

GROSS PATHOLOGY
- All animals from the group treated at 5 % were observed with scabbing (minimal/moderate) at the application site at necropsy.
- No other treatment-related findings were observed at necropsy.

- For more details, refer attached PDF document titled 'Figures and tables'

Dose descriptor:

NOEL

Remarks:

local effect

Effect level:

2 other: %

Based on:

test mat.

Sex:

female

Basis for effect level:

other: At 5 %, minimal irritation characterised by slight to well-defined erythema and oedema were noted from Day 6 onwards. At necropsy, scabbing (minimal/moderate) was noted in all animals treated with 5 %. No effects were observed at 2 %.

Dose descriptor:

NOEL

Remarks:

systemic effect

Effect level:

> 5 other: %

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no treatment related-effects were observed on mortality, clinical signs, bodyweight gain and food consumption

Critical effects observed:

not specified

None

Conclusions:

Under the test conditions, the No Observed Effect Levels (NOEL) for local and systemic effects for Mexoryl SAB to female rats were considered to be 2 and > 5 %, respectively.

Executive summary:

In a repeated dose dermal toxicity study conducted similarly to the OECD Guideline 410 and in compliance with GLP, groups of five virgin female rats of the Crl:CD (SD) BR (VAF plus) strain were dosed once daily for 10 days by dermal application with suspensions of Mexoryl SAB at dose levels of 2 and 5 %, using a dose volume of 2 mL/kg bw. A similar group of females was dosed with the vehicle (PEG 300) only, and served as controls. The formulations were applied directly to the skin of a clipped area of the back (not less than 10 % of the body area) and, approximately 6 h after application, the test site was washed using warm water and dried. Examinations during the study included: mortality, clinical observation, body weight change, evaluation of cutaneous tolerance and macroscopic necropsy.

No mortality was recorded during the study. Animals treated at 5 % were observed to be scratching at the application site from Day 8 of the dosing period onwards. In this group, very slight erythema was noted in 2-5 animals from Day 6 onwards and well-defined erythema was noted in one female between Days 8 and 10 of the dosing period. In addition, between 3 and 4 animals from this group were observed with very slight oedema from Day 7 of the dosing period. No treatment-related changes were observed in food consumption and bodyweight gain. Occasionally, transient weight losses were observed which, because of the small group sizes, skewed the means. All animals from the group treated at 5 % were observed with scabbing (minimal/moderate) at the application site at necropsy. No other treatment-related findings were observed at necropsy.

Under the test conditions, the No Observed Effect Levels (NOEL) for local and systemic effects for Mexoryl SAB to female rats were considered to be 2 and > 5 %, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information as a whole meets the tonnage driven data requirement of REACH. Moreover, reliability and consistency are observed across the different studies.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 June – 28 July, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Preliminary study performed in GLP laboratory similarly to OECD Guideline 410, used as a range-finder experiment for an OECD Guideline 414 study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

tested at 2 dose levels only; duration of exposure: 10 days; only female animals were used; haematological tests, clinical biochemistry tests and detailed histopathological examinations were not followed

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)BR (VAF plus)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK. Ltd., Margate, England
- Weight at study initiation: 200-300 g
- Housing: Housed individually in grid-bottomed polypropylene cages
- Diet (e.g. ad libitum): Pelleted diet SQC Rat and Mouse No. 1 (expanded) (Special Diets Services Limited, Witham, Essex, U.K.), ad libitum
- Water (e.g. ad libitum): Tap water (in bottles), drawn directly from the water mains, ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 40-69 %
- Air changes: 16 air changes/h
- Photoperiod: 12 h dark/ 12 h fluorescent light

Type of coverage:

open

Vehicle:

other: PEG 300

Details on exposure:

TEST SITE
- Area of exposure: Clipped area of the back
- % coverage: Not less than 10 % of the body area
- Type of wrap if used: Test article formulations were applied directly to the skin of the clipped area of the back.
- Time intervals for shavings or clipplings: Approximately 24 h before the start of dosing, fur (equivalent to not less than 10 % of the surface area of the body) was removed from the dorsal area by clipping or shaving. During the dosing period, the application sites were clipped as often as was necessary to maintain them free of fur.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Test sites were washed using warm water to remove residual test article and the skin was then blotted dry with a paper towel.
- Time after start of exposure: Approximately 6 h

TEST MATERIAL
- Constant volume or concentration used: Yes, 2 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, animals were fitted with Elizabethan collars for the 6 h exposure period in order to prevent ingestion of the test article.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

6 h/day; 10 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 2 and 5 %
Basis:
other: directly applied on skin

No. of animals per sex per dose:

Five virgin females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Selected females were allocated randomly to treatment group using a stratified bodyweight procedure.

Positive control:

No

Observations and examinations performed and frequency:

MORTALITIES:
- Time schedule: Twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded over Days 1-4, 4-7 and 7-10 of the study

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Daily

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all surviving animals were killed by CO2 asphyxiation at the end of treatment period and were submitted to the procedure of necropsy including the following examinations: appearance of tissues in situ from the cranial, thoracic and abdominal cavities.
HISTOPATHOLOGY: Treated skin and a section of control skin (taken from the right hind limb) from all animals was fixed and stored in neutral buffered formaldehyde. These tissues were not examined microscopically.

Other examinations:

None

Statistics:

Analysis of variance (ANOVA) was performed on all parameters, using treatment group as the factor in the analysis. Residuals from this preliminary analysis were examined for heterogeneity of variance using Levenes test. If Levenes test was significant at the 1 % level, then the particular variable concerned was subjected to a non-parametric analysis. Otherwise, William’s test was performed to compare the high dose with control at the two-sided 5 % level. If this test was statistically significant then comparisons of the subsequent doses against control was performed at the one-sided 5 % level until a non-significant difference was found, at which point the testing stopped. The highest dose at which the difference from control was non-significant was deemed to be the no-effect level.
If Levenes test indicated that there were significant differences in the treatment group variances or if a parametric analysis was deemed to be inappropriate, then a Kruskal-Wallis ANOVA was performed to assess overall differences between the treatment groups, followed by Shirley's nonparametric version of William’s test, which is based on mean ranks rather than the arithmetic means.

Clinical signs:

effects observed, treatment-related

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
- No premature death was noted.
- Animals from the group treated at 5 % were observed to be scratching at the application site from Day 8 of the dosing period onwards.
- No other treatment-related clinical observation was noted.

DERMAL IRRITATION:
- Between 2 and 5 animals from the group treated at 5 % were observed with very slight erythema from Day 6 onwards and one female was observed with well-defined erythema between Days 8 and 10 of the dosing period. In addition, between 3 and 4 animals from this group were observed with very slight oedema from Day 7 of the dosing period. There were no skin reactions in either of the other groups.

BODY WEIGHT AND WEIGHT GAIN:
- No effect of treatment was noted on bodyweight gain throughout the dosing period.
- Occasionally, transient weight losses were observed which, because of the small group sizes, skewed the means.

FOOD CONSUMPTION:
- No effect of treatment was noted on mean food consumption.

GROSS PATHOLOGY
- All animals from the group treated at 5 % were observed with scabbing (minimal/moderate) at the application site at necropsy.
- No other treatment-related findings were observed at necropsy.

- For more details, refer attached PDF document titled 'Figures and tables'

Dose descriptor:

NOEL

Remarks:

local effect

Effect level:

2 other: %

Based on:

test mat.

Sex:

female

Basis for effect level:

other: At 5 %, minimal irritation characterised by slight to well-defined erythema and oedema were noted from Day 6 onwards. At necropsy, scabbing (minimal/moderate) was noted in all animals treated with 5 %. No effects were observed at 2 %.

Dose descriptor:

NOEL

Remarks:

systemic effect

Effect level:

> 5 other: %

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no treatment related-effects were observed on mortality, clinical signs, bodyweight gain and food consumption

Critical effects observed:

not specified

None

Conclusions:

Under the test conditions, the No Observed Effect Levels (NOEL) for local and systemic effects for Mexoryl SAB to female rats were considered to be 2 and > 5 %, respectively.

Executive summary:

In a repeated dose dermal toxicity study conducted similarly to the OECD Guideline 410 and in compliance with GLP, groups of five virgin female rats of the Crl:CD (SD) BR (VAF plus) strain were dosed once daily for 10 days by dermal application with suspensions of Mexoryl SAB at dose levels of 2 and 5 %, using a dose volume of 2 mL/kg bw. A similar group of females was dosed with the vehicle (PEG 300) only, and served as controls. The formulations were applied directly to the skin of a clipped area of the back (not less than 10 % of the body area) and, approximately 6 h after application, the test site was washed using warm water and dried. Examinations during the study included: mortality, clinical observation, body weight change, evaluation of cutaneous tolerance and macroscopic necropsy.

No mortality was recorded during the study. Animals treated at 5 % were observed to be scratching at the application site from Day 8 of the dosing period onwards. In this group, very slight erythema was noted in 2-5 animals from Day 6 onwards and well-defined erythema was noted in one female between Days 8 and 10 of the dosing period. In addition, between 3 and 4 animals from this group were observed with very slight oedema from Day 7 of the dosing period. No treatment-related changes were observed in food consumption and bodyweight gain. Occasionally, transient weight losses were observed which, because of the small group sizes, skewed the means. All animals from the group treated at 5 % were observed with scabbing (minimal/moderate) at the application site at necropsy. No other treatment-related findings were observed at necropsy.

Under the test conditions, the No Observed Effect Levels (NOEL) for local and systemic effects for Mexoryl SAB to female rats were considered to be 2 and > 5 %, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

167 µg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information as a whole meets the tonnage driven data requirement of REACH. Moreover, reliability and consistency are observed across the different studies.

Additional information

In a 4 week oral toxicity study performed in accordance with OECD guideline 407 and in compliance with GLP, Mexoryl SAB was administered through gavage at dose levels of 0, 10, 30, 100 and 300 (then 200) mg/kg bw/day for 4 weeks. 2 recovery groups (for control and the highest dose groups) were maintained undosed for further 14 days after the 4-week treatment. The dose level of the high dose group was dropped from 300 to 200 mg/kg bw/day on day 13 of the treatment period because of adverse clinical signs, including death. There were 5 deaths in the highest dose group during treatment. Rough coat, piloerection and post dose salivation were noted in the highest dose group. Bodyweight gain was reduced slightly (13-14 %) for males of the highest dose group during the treatment and treatment-free periods and for females given 100 mg/kg bw/day during the treatment period only. Males given the highest dose had reduced food consumption during both the treatment and treatment-free period. Haematology and blood chemistry examinations did not reveal any effects. Macroscopic observations at necropsy revealed a dose-related increased incidence of stomach abnormalities for animals given 100 and 300/200 mg/kg bw/day at the end of both the treatment and treatment-free period. Microscopic pathology revealed hyperplasia of the non-glandular stomach for animals given 300/200 mg/kg bw/day, accompanied by chronic inflammation and ulceration. One male given 100 mg/kg bw/day also showed hyperplasia. Similar but less severe hyperplastic lesions were also observed for the recovery animals previously given 300/200mg/kg bw/day. The hyperplasia of the non glandular mucosa of the stomach was less severe at the end of the treatment-free period indicating that some recovery had taken place.

Therefore, the results showed severe local effects in the non-glandular stomach of animals administered 300/200 mg/kg bw/d and at 100 mg/kg bw/d with less severity (one male). At lower doses (10 and 30 mg/kg bw/d), no local effects were observed. No systemic effect was observed at any doses up to the highest dose bearable for the animals corresponding to 100 mg/kg bw/d, i.e. without severe non-glandular irritation leading to death. Hence, NOEL for local effect was 30 mg/kg bw/d while NOEL for systemic effect was higher than 100 mg/kg bw/d considered as the highest dose bearable for the animals (i.e. without mortality or without severe gastro-intestinal tract irritation).

Under the test conditions of this study, NOEL for local effect was 30 mg/kg bw/d and the NOEL for systemic effect was higher than 100 mg/kg bw/d. This dose was considered as the highest dose exposure that it was ethically acceptable to use.

In a repeated dose dermal toxicity study conducted similarly to the OECD Guideline 410 and in compliance with GLP, groups of five virgin female rats of the Crl:CD (SD) BR (VAF plus) strain were dosed once daily for 10 days by dermal application with suspensions of Mexoryl SAB at dose levels of 2 and 5 %, using a dose volume of 2 mL/kg bw. A similar group of females was dosed with the vehicle (PEG 300) only, and served as controls. The formulations were applied directly to the skin of a clipped area of the back (not less than 10 % of the body area) and, approximately 6 h after application, the test site was washed using warm water and dried. Examinations during the study included: mortality, clinical observation, body weight change, evaluation of cutaneous tolerance and macroscopic necropsy.

No mortality was recorded during the study. Animals treated at 5 % were observed to be scratching at the application site from Day 8 of the dosing period onwards. In this group, very slight erythema was noted in 2-5 animals from Day 6 onwards and well-defined erythema was noted in one female between Days 8 and 10 of the dosing period. In addition, between 3 and 4 animals from this group were observed with very slight oedema from Day 7 of the dosing period. No treatment-related changes were observed in food consumption and bodyweight gain. Occasionally, transient weight losses were observed which, because of the small group sizes, skewed the means. All animals from the group treated at 5 % were observed with scabbing (minimal/moderate) at the application site at necropsy. No other treatment-related findings were observed at necropsy.

Under the test conditions, the No Observed Effect Levels (NOEL) for local and systemic effects for Mexoryl SAB to female rats were considered to be 2 and > 5 %, respectively.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study performed according to OECD 407 guideline and GLP.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Key study performed according to OECD 407 guideline and GLP.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Preliminary study performed in rats exposed by dermal route for 10 days considered as key study to observe local effects.

Justification for classification or non-classification

In a 28-day repeated oral toxicity study, macroscopic observations at necropsy revealed a dose-related increased incidence of stomach abnormalities for animals given 100 and 300/200 mg/kg bw/day at the end of both the treatment and treatment-free period. Microscopic pathology revealed hyperplasia of the non-glandular stomach for animals given 300/200 mg/kg bw/day, accompanied by chronic inflammation and ulceration. One male given 100 mg/kg bw/day also showed hyperplasia. Similar but less severe hyperplastic lesions were also observed for the recovery animals previously given 300/200mg/kg bw/day. These effects may be due to the irritant properties of the substance and the mode of administration of the test substance (oral gavage). Also, reversibility was shown and the effects are limited to the non-glandular stomach, therefore they are of doubtful relevance to humans.

NOEL for local effect was 30 mg/kg bw/d and NOEL for systemic effect was higher than 100 mg/kg bw/d considered as the highest dose exposure that it was ethically acceptable to use.

Thus, Mexoryl SAB is not classified according to the Regulation (EC) No. 1272/2008 (CLP) and the Directive 67/548/EEC.