2-hydroxybenzoic acid 2-butyloctyl ester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a 28-day oral gavage study with the subject materail there were no adverse effects noted on reproductive organs.

Link to relevant study records

Reference

Endpoint:

three-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

To address some of the toxicological endpoints as part of the REACH registration of 2-Hydroxybenzoic acid, 2-Butyloctyl ester (CAS 190085-41-7) (target substance), it is proposed to read across to methyl salicylate (CAS 119-36-8) (source substance) to fulfill the data requirements of the OECD 416 study.
Both the above mentioned substance share similar chemical structures. As mentioned, both substances have identical functionality, they are both esters of salicylic acid. The only difference in the two substances is in the alcohol portion of the ester group.
Read across has been deemed as suitable.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

several deficiencies in relation to OECD Guideline 416 in terms of parameters studied

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet: ad libitum (Purina Laboratory Chow)
- Water: ad libitum
- Acclimation period: no data


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


IN-LIFE DATES: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): the diet was prepared every 14 days in a manner identical to that of Webb and Hansen (1963) and according to the results of Jones et al (1962).
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

VEHICLE: none

Details on mating procedure:

no information available

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

no information available

Duration of treatment / exposure:

100 days prior to the first mating until study termination

Frequency of treatment:

continuous in the feed

Details on study schedule:

no information available

Remarks:

Doses / Concentrations:
0, 500, 1500, 3000 and 5000 ppm
Basis:
nominal in diet
(equivalent to 25, 75, 150, 250 mg/kg bw as Methyl Salicylate, or 22.5, 67.5, 135, 225 mg/kg bw as Salicylic Acid)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

no information available

Positive control:

none employed

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: No
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter ; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

not examined

Postmortem examinations (offspring):

SACRIFICE: no data

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for the third generation only.

HISTOPATHOLOGY :
microscopic examination of livers and kidneys was performed.

Statistics:

The Chi-square test was used to determine significant differences between each dose and the control for each mating in each generation.

Reproductive indices:

The fertility index (number of litters/number of females mated).

Offspring viability indices:

viability index (number of liveborn/total number born)
survival index (number alive at day 4/ number born alive)
weaning index (adjusted number of day 21 survivors/number alive at day 4)

Clinical signs:

no effects observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

- Fertility index: no significant differences for any dose in the 1st generation. Appreciable decreases seen in the 2nd and 3rd generations at the highest dose level (5000 ppm).
- Average litter size/female: significant decreases were seen in the second generation in the second mating at 3000 ppm and in both matings at 5000 ppm. Although decreases were seen at 1500 ppm, they were not statistically significant because of the large variation in progeny between females within a group.

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects at the highest dose tested

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects at the highest dose tested

Remarks on result:

other: Generation: reproduction (migrated information)

Dose descriptor:

LOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced neonate viability

Remarks on result:

other: Generation: development (migrated information)

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced neonate viability

Remarks on result:

other: Generation: development (migrated information)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)

- The average number of liveborn young per mated female: Statistically significant differences were observed in both matings of the second generation at 3000 ppm and at 5000 ppm.

- Viability index: "possible loss of young through stillbirths" in 2 matings/5000 ppm.

- Average no. of surviving progeny/female, day 4: significant decreases occurred in both matings of the second generation at 3000 ppm and 5000 ppm

- Survival index, day 4: an adverse effect was observed in the second generation at the 3000 and 5000 ppm and in the first mating of the third generation at the same dose levels.

- Average no. progeny weaned/female, day 21: significant decreases were observed in the second generation at 3000 ppm in the first mating and at 5000 ppm in the first and second matings.

- Weaning index: "appreciable decrease" in 2nd generation/2nd litter/5000 ppm.

BODY WEIGHT:

- Average weanling weight,( day 21/sex): decreases in weight appeared consistently at the 3000 and 5000 ppm levels(in all generations).

GROSS PATHOLOGY (OFFSPRING)
no grossly visible abnormalities.

HISTOPATHOLOGY (OFFSPRING)
Histopathological examinations of the livers and kidneys of 3rd weanlings at the 0, 3000 and 5000 ppm dose levels showed no indication of toxic effects.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 500 ppm (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

not specified

Supplemental study: the results obtained after addition of calcium carbonate to methyl salicylate did not differ from those obtained after administration of methyl salicylate alone.

Table 1 : fertility indexes of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0		500		1500		3000		5000
generation	mating	FI (a)	% (b)	FI	%	FI	%	FI	%	FI	%
1	1	20/20	100	20/20	100	20/20	100	20/20	100	20/20	100
	2	19/19	100	20/20	100	18/19	95	19/19	100	20/20	100
2	1	20/20	100	19/20	95	20/20	100	19/20	95	17/20	85
	2	19/19	100	19/20	95	19/19	100	19/20	95	10/13	77
3	1	20/20	100	18/20	90	18/19	95	19/20	95	17/19	89
	2	18/20	90	16/18	89	17/19	89	15/17	88	16/19	84

(a) Fertility Index (number of litters/ number of females mated)

(b) Percent females pregnant

Table 2: average litter size of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0		500		1500		3000		5000
generation	mating	No. (a)	Av. (b)	No.	Av.	No.	Av.	No.	Av.	No.	Av.
1	1	208/20	10.4	211/19	11.1	207/20	10.4	235/20	11.8	188/18	10.4
	2	213/19	11.2	232/20	11.6	228/19	12.0	238/19	12.5	198/19	10.4
2	1	216/20	10.8	205/20	10.2	206/20	10.3	169/20	8.4	124/20	6.2 (c)
	2	226/19	11.9	204/20	10.2	189/18	10.5	187/20	9.4 (d)	86/13	6.6 (c)
3	1	192/20	9.6	188/19	9.9	172/19	9.1	170/20	8.5	179/19	9.4
	2	197/20	9.8	191/18	10.6	163/19	8.6	132/17	7.38	172/19	9.1

(a) Total number progeny/number females mated

(b) Average litter size per female mated

(c) significant at P<0.01

(d) significant at P<0.05

Table 3: viability data for rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)
1	1	208/20	10.4	1,00	211/19	11.1	1,00	195/20	9,8	0,94	229/20	11,4	0,97	167/18	9,3	0,88
	2	213/19	11.2	1,00	231/20	11.6	1,00	226/19	11,9	0,99	237/19	12,5	1,00	189/19	9,9	0,95
2	1	215/20	10.8	1,00	203/20	10.2	0,99	203/20	10,2	0,99	164/20	8,2 (e)	0,97	106/19	5,6 (f)	0,85
	2	225/19	11.8	1,00	203/20	10.2	1,00	189/18	10,5	1,00	182/20	9,1 (e)	0,97	82/13	6,3 (f)	0,95
3	1	188/20	9,4	0,98	184/19	9,7	0,98	160/19	8,4	0,93	164/20	8,2	0,96	174/19	9,2	0,97
	2	196/20	9.8	1,00	186/18	10,3	0,97	155/19	8,2	0,95	118/17	6,9	0,89	166/19	8,7	0,97

(a) Total number liveborn/number females mated

(b) Average number liveborn per female mated

(c) Viability index (no. liveborn/total no. born)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

Table 4: survival data of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	SI (c, d)	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI
1	1	157/17	9,2	0,90	116/14	8,3	0,82	172/19	9,1	0,96	152/15	10,1	0,92	129/15	8,6	0,94
	2	202/19	10,6	0,95	196/20	9,8	0,85	205/19	10,8	0,91	218/19	11,5	0,92	168/19	8,8	0,89
2	1	188/20	9,4	0,87	179/20	9	0,88	190/20	9,5	0,94	123/20	6,2 (e)	0,75	82/19	4,3 (f)	0,77
	2	211/19	11,1	0,94	188/20	9,4	0,93	186/18	10,3	0,98	165/20	8,2 (e)	0,91	61/13	4,7 (f)	0,74
3	1	174/20	8,7	0,93	177/19	9,3	0,96	147/19	7,7	0,92	139/20	7	0,85	147/19	7,7	0,84
	2	174/20	8,7	0,89	179/18	9,9	0,96	150/19	7,9	0,97	113/17	6,6	0,96	153/19	8,1	0,92

(a) Total number day 4 survivors / no. females mated

(b) Average number day 4 survivors per female mated

(c) Survival index (no. day 4 survivors/total no. liveborn)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

See "Overall remarks, attachments" for Table 5.

Conclusions:

Under the conditions of this study, methyl salicylate did not significantly reduce male or female fertility. Methyl salicylate did induce developmental toxicity: adverse effects on offspring viability were observed but with no evidence of increased incidence of malformations at any dose tested.
The NOAELs were identified:
NOAEL (parental): 250 mg/kg bw/day
NOAEL (reproduction): 250 mg/kg bw/day
LOAEL (development): 150 mg/kg bw/day
NOAEL (development): 75 mg/kg bw/day

Executive summary:

In this 3 -generation study, rats were fed methyl salicylate at doses of 500, 1500, 3000 or 5000 ppm in the diet (equivalent to 25, 75, 150 or 250 mg/kg body weight as methyl salicylate, or 22.5, 67.5, 135, 225 mg/kg bw as salicylic acid) 100 days before the first mating and then throughout the experiment. No clinical signs of toxicity were reported at any dose level. No statistically significant decrease was reported in fertility indexes at any dose level at the F1 generation, however it was considered that there were "appreciable" decreases at 250 mg/kg in the F2 and F3 generations. Significant decreases were reported in average litter size, average number of live-born progeny, average numbers of survivors to PND4 and average number of weaning in the 150 and 250 mg/kg bw/day in the F2 generation. No external malformations were reported in pups of any litter and necropsy of the third generation weanlings showed no significant findings. The effects in a calcium carbonate supplement groups did not differ significantly from those of the groups fed methyl salicylate alone.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The quality of the study was judged adequate for the purpose of classification.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The metabolism of the subject material, 2 -butyloctyl salicylate, and other salicylic esters have been investigated (Belsito et al., 2007). These salicylate esters were found to be rapidly and completely metabolised by esterases in the skin, plasma, liver and other body tissues to salicylic acid and the corresponding alcohol, here 2 -octylhexyl alcohol. Although information is lacking, the alcohol metabolite, 2 -hexyloctyl alcohol, should be rapidly and completely metabolized and eliminated and will not contribute to the toxicological hazard of the subject material (OECD, 2006). Salicylic acid is rapidly and completely eliminated in a number of species with elimination almost exclusively in the urine (Belsito et al., 2007).

In a 3-generation study, male and rats were fed methyl salicylate at doses of 500, 1500, 3000 or 5000 ppm in the diet (equivalent to 25, 75, 150 or 250 mg/kg body weight as Methyl salicylate, or 22.5, 67.5, 135, 225 mg/kg bw as salicylic acid) 100 days before the first mating and then throughout the experiment (Collins et al., 1971). No clinical signs of toxicity were reported at any dose level. No statistically significant decrease was reported in fertility index at any dose; however it was considered that there were "appreciable" decreases at 250 mg/kg in F2 and F3. Significant decreases were reported in average litter size, average number of live-born progeny, average numbers of survivors to PND4 and average number of weaning in the 150 and 250 mg/kg bw/day in F2. No external malformations were reported in pups of any litter and necropsy of the third generation weanlings showed no significant findings. Calcium carbonate supplemented groups did not differ significantly from those of the groups fed methyl salicylate alone. The following NOAELs/LOAEL were determined in this study: NOAEL (parental): 250 mg/kg bw/day; NOAEL (reproduction): 250 mg/kg bw/day; LOAEL (development): 150 mg/kg bw/day; NOAEL (development): 75 mg/kg bw/day.

A reproductive toxicity screening study conducted according to OECD Guideline 421 (Symrise, 2013), the surrogate material, 2-ethylhexyl salicylate, was administered by oral gavage at doses of 0, 25, 80 and 250 mg/kg bw/day to males for 28 days and to females for 7 weeks.
At the dose level of 250 mg/kg bw/day, one female was found dead on day 23 of the gestation period. During necropsy, fetuses were found in uterus of this female. No other test item-related findings were noted in males or females at any dose level. At dose levels of 250 and 0 mg/kg bw/day, a reduction in gestation indexes as well as increases in post-implantation losses resulting in lower litter sizes were noted. These effects were considered to be test item related. Prolonged gestations were noted in some females of the mid- and high-dose levels and post-implantation losses occurred predominantly in these females. Due to prolonged gestation, reduced gestation index and increased post-implantation loss resulting in lower litter size at the dose levels of 250 and 80 mg/kg bw/day, NOEL for reproduction toxicity was considered to be 25 mg/kg bw/day.

Short description of key information:

In a key study with the surrogate material, methyl salicylate, there was not significant reproductive toxicity reported.

Justification for selection of Effect on fertility via oral route:

In a 3-generation study in rats with the surrogate material, methyl salicylate, there were no significant effects on reproduction noted up to the highest dose level of 250 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

The surrogate material, 2-ethylhexyl salicylate, produced only reduced body weights in rat offspring from an OECD 421 reproductive screening study at a highest dose level of 250 mg/kg bw/day. No other developmental effects were noted.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 06 to November 05, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: RccHanTM: WIST(SPF)

Details on test animals or test system and environmental conditions:

Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
Number of Animals: 44 males: 11 per group 44 females: 11 per group

Age (at Start of Treatment): 11 weeks
Body Weight Range
(at Start of Treatment): Males: 312 to 351 g Females: 208 to 244 g
Identification: Cage card and individual animal number (ear tattoo).
Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Husbandry
Room Numbers, Füllinsdorf: E0441A (acclimatization) and E0441 (after acclimatization) Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of ran-domization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg/Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/ lot nos. 100099. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.

Diet: Pelleted standard Harlan Teklad 2018C (batch no. 43/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. .
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Four groups of 11 males and 11 females were treated by gavage with Neo Heliopan OS once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

Vehicle and Control Item
Identification: Corn oil
Source: Carl Roth GmbH
Batch Number: 292189296
Expiry Date (Retest Date): 02-Aug-2017
Storage Conditions: Room temperature (20 ± 5 °C)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 1 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration.
The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to B. Bürkle (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by GC coupled to an FID detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

In conclusion, the results indicate the accurate use of the test item and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

Duration of treatment / exposure:

Males: 28 days Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Duration of test:

Approximately 7 weeks

Remarks:

Doses / Concentrations:
0, 25, 80, 250 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group)
Group 2: 25 mg/kg/day
Group 3: 80 mg/kg/day
Group 4: 250 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D54872, using dose levels of 0, 100, 300 and 1000 mg/kg/day, resulting in mortality and adverse toxic effects at the dose level
of 1000 mg/kg bw/day and adverse toxic effects at the dose level of 300 mg/kg bw/day.
Dose Volume: 4 mL/kg body weight
Dose Concentrations: Group 1: 0.00 mg/mL
Group 2: 6.25 mg/mL
Group 3: 20.00 mg/mL
Group 4: 62.50 mg/mL
Duration of Acclimatization Period: Minimum 5 days
Duration of Treatment Period: Males: 28 days Females: Approximately 7 weeks

Maternal examinations:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
Females: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 and days 1 - 4 of the lactation.
No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.

Ovaries and uterine content:

Ovaries were checked for discoloration and uteri for fetus content and discoloration.

Fetal examinations:

Pup Data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum. Pups and dams were sacrificed on day 4 post partum. All parent animals and pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

• Means and standard deviations of various data were calculated.

• The Dunnett-test [see References (2)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test [see References (3)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test [see References (4)] was applied if the variables could be dichotomized without loss of information.

Indices:

Reproductive indices recorded were fertility indices, mean precoital time, post-implantation loss, and offspring viability indices were mean litter size, pup sex ratios and viability indices.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At 250 mg/kg bw/d slight but non-significant changes on weight gain were noted.

Key result

Dose descriptor:

NOEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No test item-related effects on mating performance, fertility index (number of females achieving pregnancy as a percentage of females paired), conception rate (number of females achieving pregnancy as a percentage of females mated), corpora lutea count, number of implantations or postnatal loss were noted at any dose level.
Treatment with the test item at the dose levels of 250 and 80 mg/kg bw/day caused a reduction in gestation index (number of females with living pups as a percentage of females pregnant) as well as an increase in incidence of post-implantation loss resulting in a lower litter size. Further, at the dose levels of 250 and 80 mg/kg bw/day, prolonged gestation period was noted. These findings were considered to be test item-related adverse effects.

Key result

Dose descriptor:

NOEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

not specified

1 Viability / Mortality

At the dose level of 250 mg/kg bw/day, one female (no. 78) was found dead on day 23 of the gestation period. During necropsy, fetuses were found in uterus of this female. A slight body weight loss was noted in this female on day 22 of the gestation period but no other signs and no macroscopical or microscopical findings indicating bad condition of this female were noted. Death of the female was most probably a result of difficult parturition and considered to be test-item related. All remaining animals of P generation survived the scheduled study period.

2 Clinical Signs or Observations

No test item-related findings were noted in males or females at any dose level. In one female (67) at the mid-dose level, hunched posture and ruffled fur were noted from day 2 of the lactation period onwards. Further, slight swelling in axillary region was observed in one female (no. 79) at the high-dose level from day 9 of the gestation period onwards. Because of isolated occurrence, these findings were considered not to be related to the treatment with the test item. No further findings were noted in males or females at any dose level.

3 Food Consumption of Males

The overall differences in mean food consumption at the dose levels of 25, 80 and 250 mg/kg bw/day were respectively: +0.9%, -0.9% and +1.7% during the pre-pairing period (percentages refer to the respective values in the control group).

4 Food Consumption of Females Pre-Pairing, Gestation and Lactation Periods

At the dose levels of 250 and 80 mg/kg bw/day, reduced food consumption was noted during lactation. Mean food consumption was 16.4 and 19.8 g/animal/day at the dose levels of 250 and 80 mg/kg bw/day, respectively and 22.2 g/animal/day in the control group. Although the differences were not statistically significant, they were dose dependent and therefore considered to probably be related to the treatment with the test item. No effects on food consumption were noted at the dose level of 80 mg/kg bw/day. The overall differences in mean food consumption at the dose levels of 25, 80 and 250 mg/ kg bw/day were respectively: -5.6%, -0.6% and -4.4% during the pre-pairing period, -2.4%, +6.2% and +4.8% during the gestation period and +1.4%, -10.8% and -26.1% during the lactation period (percentages refer to the respective values in the control group).

5 Body Weights of Males Pre-Pairing and Pairing Periods

At the dose level of 250 mg/kg bw/day, slight but statistically significant reduction in body weight gain was noted on day 13 of the pre-pairing period. Body weight gain was also slightly lower on further days at the end of this period but without statistical significance. No significant differences in absolute body weights were noted at this dose level. At the dose levels of 25 and 80 mg/kg bw/day, no test item-related effects on absolute body weights or body weight gain were noted. The overall differences in mean body weight gain at the dose levels of 0, 25, 80 and 250 mg/kg bw/day were respectively: +15%, +15%, +13% and +13% during the pre-pairing period and +11%, +9%, +9% and +10% during the pairing period (percentages refer to the body weight gain within the period). At the low- and mid-dose levels, statistically significantly lower body weight gains were noted on individual days during the pairing period. Because the differences were minor and did not follow dose dependency, they were considered not to be related to the treatment with the test item. 6 Body Weights of Females Pre-Pairing, Pairing, Gestation and Lactation Periods At the dose level of 250 mg/kg bw/day, statistically significant reduction in body weight gain was noted on day 4 of the lactation period. No significant differences in absolute body weights were noted at this dose level at any time. At the dose levels of 25 and 80 mg/kg bw/day, no effects on absolute body weights or body weight gain were noted. The overall differences in mean body weight gain at the dose levels of 0, 25, 80 and 250 mg/kg bw/day were respectively: +8%, +7%, +9% and +6% during the pre-pairing period, +51%, +55%, +56% and +48% during the gestation period and +5%, +2%, +2% and ±0% during the lactation period (percentages refer to the body weight gain within the period). Mating Performance and Fertility: No effects on mating performance, fertility index or conception rate were noted at any dose level. No effects on gestation index were noted at the dose level of 25 mg/kg bw/day; it was 100% at this dose level. Duration of Gestation : At the dose levels of 250 and 80 mg/kg bw/day, prolonged gestation period was noted. Mean duration of gestation was 22.6 and 22.0 days at the dose levels of 250 and 80 mg/kg bw/day, respectively, compared to 21.5 days in the control group. Mean prolongation of the gestation at the high- and mid-dose levels was not statistically significant. However, it was dose dependent and the values were beyond the biological background (historical control data included values of gestation length from 21.2 to 21.8 days). Therefore this finding was considered to be test item-related. At the dose level of 25 mg/kg bw/day, mean gestation of duration was the same like the control value; 21.5 days and therefore not affected by the treatment. Corpora Lutea Count : No effects on corpora lutea count were observed at any dose level. Implantation Rate and Post-Implantation Loss : Number of implantations was not affected by the treatment with the test item at any dose level. At the dose level of 25 mg/kg bw/day, no effects on post-implantation loss were noted.

Conclusions:

Because of reduced absolute body weights of pups at the dose level of 250 mg/kg bw/day NOEL for developmental toxicity was considered to be 80 mg/kg bw/day.

Executive summary:

Because of reduced absolute body weights of pups at the dose level of 250 mg/kg bw/day NOEL for developmental toxicity was considered to be 80 mg/kg bw/day. No test item-related observations were noted in pups during the first litter check or during lactation at any dose level. Pups sex ratio was not affected by the exposure to the test item at any dose level. Treatment with the test item at the dose level of 250 mg/kg bw/day caused a reduction in body weights of pups recorded on day 1 and 4 of the lactation period. During this period, body weight gain of pups at the high-dose level was similar to body weight gain of pups in the control group. Reduction in absolute body weights of pups was considered to be test item-related adverse effect. No test item-related effects on body weights or body weight gain of pups were noted at the dose levels of 25 and 80 mg/kg bw/day. No test item-related macroscopical findings were found in pups at any dose level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The quality of the study was judged adequate for purposes of classification.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD guideline 421 study in rats, the test item, 2 -ethylhexyl salicylate, was administered in corn oil as vehicle at dosages of 25, 80, and 250 mg/kg body weight/day, and controls received the vehicle only (Symrise, 2013). The test article was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Because of reduced absolute body weights of pups at the highest dose level of 250 mg/kg bw/day, the NOEL for developmental toxicity was considered to be 80 mg/kg bw/day. No test item-related observations were noted in pups during the first litter check or during lactation at any dose level. The reduction in absolute body weights of pups was considered to be a test item-related adverse effect. No test item-related effects on body weights or body weight gain of pups were noted at the dose levels of 25 and 80 mg/kg bw/day. No test item-related macroscopical findings were found in pups at any dose level.

Justification for selection of Effect on developmental toxicity: via oral route:

For the surrogate material, 2-ethylhexyl salicylate, only reduced pup body weights in rats were observed at the hghest dose level tested in a guideline OECD 421 reproductive effects screening study. No other pathological effects were reported.

Toxicity to reproduction: other studies

Additional information

Supporting information from a 28 -day oral gavage study in rats on the subject material further supports the lack of reproductive toxicity. The subject material was administered orally by gavage to Sprague-Dawley CD rats (5/sex/group) at dose levels of 15, 150 or 1000 mg/kg bw/day. There were no gross pathological or histopathological changes noted in the reproductive organs of any treated animals.

Justification for classification or non-classification

Fertility: 

Not classified for effects on reproduction (fertility) according to CLP (Regulation EC No 1272/2008). Based primarily on information for the surrogate material, methyl salicylate. When tested in a 3-generation study, methyl salicylate and thus also the metabolite, salicylic acid, did not adversely affect fertility.

Development: 

Not classified for effects on reproduction (development) according to CLP (Regulation EC No 1272/2008). Based on evidence from animal studies and human data on acetylsalicylic acid, as discussed fully in the publically available REACH dossier on methyl salicylate, different species show a variation in sensitivity to the developmental toxicity of salicylates, including salicylic acid. The rat is the most sensitive species, demonstrating effects which might lead to classification. On the other hand, salicylates do not induce developmental effects in the rabbit even at doses causing severe maternal toxicity. An extensive analysis of data on acetylsalicylic acid in human pregnancy indicates that humans are relatively insensitive, allowing the conclusion that salicylic acid should not be considered a developmental toxicant in humans. Classification is therefore not required.