Sodium 2-mercaptoethanolate

Endpoint summary

Description of key information

 A NOAEL of 19.22 mg/kg bw/day was determined based on read-across data from CAS 60 -24 -2

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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:

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

Version / remarks:

July 27, 1995

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: ATOFINA
- Lot/batch No. of test material: 15/10/02
- Appearance: Colourless liquid
- Expiration date of the lot/batch: December 2003
- Purity: 99.852%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and under nitrogen atmosphere.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age/Weight: at the beginning of the treatment period, the males were approximately 9 weeks old and had a mean body weight of 331 g, the females were 6 weeks old and had a mean body weight of 184 g. The animals were sexually mature at the time of mating and the females were virgin.
Acclimation: an 11-day acclimation period to the conditions of the study preceded the beginning of the treatment period. A larger number of animals than necessary was acclimated to permit selection and/or replacement of individuals.
Allocation to study: before the beginning of the treatment period, the required number of animals (40 males and 60 females) was selected according to body weight and clinical condition and allocated to the groups, according to a stratification procedure, so that the average body weight of each group was similar. Identification: each animal was individually identified by an ear tattoo and received a unique CIT identity number.
From arrival at CIT, the animals were housed in a barriered rodent unit, under specific pathogen free (SPF) standard laboratory conditions.
The animal room conditions are set as follows: temperature: 22 +/- 2°C, relative humidity : 50 +/- 20%, light/dark cycle: 12h/12h (7:00 - 19:00), ventilation: about 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are checked regularly and records filed. The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.
The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage.
The F0 females were housed individually in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Autoclaved wood shavings (SICSA, Alfortville, France) were provided as nesting material, a few days before delivery and during the lactation period. The cages were placed in numerical order on the racks. Every 6 weeks, all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
The animals had free access to pelleted maintenance diet distributed weekly. The animals had free access to bottles containing tap water (filtered with a 0.22 um filter).
Bacterial and chemical analyses of sawdust, diet and water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (sawdust: pesticides and heavy metals; diet and water: pesticides, heavy metals and nitrosamines). No contaminants were present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on oral exposure:

The test item was administered as a solution in the vehicle. The test item dosage forms were prepared at weekly intervals and stored at +4°C and protected from light prior to use. Due to physical and chemical properties of the test item, special care was taken in order to ensure minimal dispersion into the environment and exposure of the technicians.
Before the start of treatment, the suitability of the proposed dosage form preparation procedure was determined. During the treatment period, the concentration of dosage forms prepared for use in the study was checked.
Before the start of treatment, two dosage forms containing 3 and 15 mg/mL of test item were prepared for stability analysis. Each dosage form was sampled in duplicate after 0 (just after preparation), 4 and 9 days storage at +4°C and protected from light. The aliquot sampled on day 4 was stored frozen at -20°C pending analysis on the last sampling occasion (day 9) when all samples were assayed.
The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1, 4, 8 and 12 was determined.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

ca. 7 weeks

Frequency of treatment:

once daily; 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 (main groups); 5 (satellite groups)

Control animals:

yes, concurrent vehicle

Details on study design:

The dose-levels were determined in agreement with the Sponsor, following the results of a preliminary 2-week toxicity study by oral route in rats (CIT/Study No. 24603 TSR). The test item, when administered daily for 2 weeks was well tolerated at 10 and 30 mg/kg/day. At 100 mg/kg/day, treatment-related changes were noted resulting in:
- the death of a few animals,
- decrease in body weight and food consumption in females,
- increase of coagulation factors and transaminase activity in males and females,
- increase urea and decrease sodium blood level in females,
- increase in the weight and the size of the liver in males and females.
Consequently, the dose-levels of 15, 50 and 75 mg/kg/day were selected.
The animals were allocated in 4 main groups (10 males and 10 females) and 4 satellite groups (5 females). The reprotoxicity end-points were evaluated in the male and female animals from the main groups. The repeated dose toxicity end-points were evaluated in the five first males from the main groups and the five females of the satellite groups.

Observations and examinations performed and frequency:

Each animal was observed at least once a day, at approximately the same time for the recording of clinical signs.
All animals of each group were observed in the cage, in the hand and in the standard arena, by observers unaware of the animal’s treatment, before the first day of treatment and then once a week thereafter.
The animals were randomized in order to ensure "blind" evaluation, except for examination performed before the first day of treatment.
The following parameters were assessed:
. "touch escape" or ease of removal from the cage,
. in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
. in the standard arena (two-minute recording): grooming, palpebral closure, defecation and urination counts, tremors, twitches, convulsions, gait, arousal (hypo- and hyperactivity),
. posture, stereotypic behavior and breathing, ataxia, hypotonia.
In the first five males of the main groups and in the five females of the satellite groups, the examinations listed below were conducted during the week before mating (before laboratory investigations). The observer performing the evaluation was not aware of the treatment group of the animal.
The animals were randomized in order to ensure "blind" evaluation. The following measurements, reflexes and responses will be recorded:
. touch response,
. forelimb grip strength,
. pupil reflex,
. visual stimulus,
. auditory startle reflex,
. tail pinch response,
. righting reflex,
. landing foot play,
. rectal temperature.
Motor activity was measured in the first five males of the main groups and in the five females of the satellite groups, during the week before mating (before laboratory investigations), using an automated infra-red sensor equipment recording individual animal activity over a 10-minute period.
The body weight of each male of the main groups and female of the satellite groups was recorded on the first day of treatment (day 1), then once a week until sacrifice. The body weight of each female of the main groups was recorded once a week during the premating and mating periods, then on days 0, 7, 14, 20 post-coitum and on days 1, 7, 14 and 21 post-partum.
The quantity of food consumed by male of the main groups and female of the satellite groups was recorded once a week, over a 7-day period, from the first day of treatment until sacrifice.
main groups.
Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food-hopper the next day.

Sacrifice and pathology:

On completion of the treatment period, all surviving animals were asphyxiated by carbon dioxide and killed by exsanguination. Any moribund animals were killed in the same way.

Statistics:

Number of corpora lutea (implantations and pups) or as proportions (pre-implantation loss, post-implantation loss and pup findings). Whenever necessary, the experimental unit of comparison was the litter. Mean quantitative values are compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Proportions are compared by Fisher exact probability test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Ptyalism noted from day 15 or 17 in 5 females of the mid dose group and in 4 females at 75 mg/kg/day; ptyalism from day 15 also in all males of the mid and high dose group and in 1 control male; no further clinical signs
- Reactivity to manipulations or to different stimuli not altered as well as the motor activity.
- The estrous cycle was not affected.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality except 1 female in the high dose group; death considered to be not treatment related (no clinical signs, slight perilobular vacuolated hepatocytes and congestion of the lung).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight decreased in males but slightly increased in TS treated females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was not altered in males; in females there was a slightly higher mean food consumption (days 1-50; correlated with body weight gain); the effect was not significant; however, it was considered to be treatment related.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Parameters not affected in males or females

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

no relevant changes were observed in females.
An altered urea level was considered to be without toxicological relevance (no effect on creatinine, no histopathological effect); effects on triglycerides (Trig) and cholesterol (Chol) were considered to be treatment related; other alterations were contributions of a few individuals and within historical range.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Parameters were unaffected by the treatment

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Liver paleness: in 1/10 males and 1/5 females of the control group, at 15 mg/kg in 1/5 females and 0/10 males, at 50 mg/kg in 4/10 males and 3/5 females, at 75 mg/kg in 6/10 males and 3/5 females;
- Accentuated lobular pattern of the liver: at 50 mg/kg in 3/10 males and in 0/5 females, at 75 mg/kg in 7/10 males and 1/5 females;
- Enlargement of the liver in 1/10 males given 75 mg/kg.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Minimal to slight hypertrophy of liver cells recorded in 2/5 females at 50 mg/kg and in 3/10 males and 4/5 females given 75 mg/kg. No treatment related effects were observed in other organs including reproductive organs.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

SEMINOLOGY
- No effect was observed on sperm motility and morphology.
- The number of testicular sperm heads was slightly (not significantly) reduced in high dose males (113.3E6 versus 121.9E6 per g of testis in controls).
- Further more epididymal sperm count was slightly decreased in all treatment groups; however, the effect was not dose dependent and/or within historical control range.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Body weight change of surviving rats (g):

Males (n = 10)		control	15 mg/kg	50 mg/kg	75 mg/kg
	days 1 - 15	+49	+43	+40	+38
	days 1 - 29	+96	+90	+73	+78
	days 1 - 50	+140	+140	+107	+124
Females (n = 5)	days 1 - 15	+47	+47	+54	+45
	days 1 - 29	+71	+82	+82	+72
	days 1 - 50	+96	+116	+113	+109

Changes in males (n = 5 per group) suggested an effect on the liver (correlated with histopathology):

	control	15 mg/kg	50 mg/kg	75 mg/kg
K+ (mmol/l)	3.97	4.02	4.36	4.67
Cl- (mmol/l)	104.1	105.5	106.9	108.6
urea (mmol/l)	3.7	4.2	7.0	7.1
creatinine (umol/l)	36	33	35	39
bile acids (umol/l)	49.8	49.4	69.8	116.7
Chol (mmol/l)	1.6	1.3	0.9	0.6
Trig (mmol/l)	0.85	0.67	0.39	0.23
ALAT (IU/l)	2	24	37	37
ASAT (IU/l)	59	61	69	134
Glucose (mmol/l)	6.72	7.29	7.16	8.25

Peri-/medio-lobular vacuolated hepatocytes:

males		0 mg/kg	15 mg/kg	50 mg/kg	75 mg/kg
	incidence	2/5	3/5	5/7	9/10
	mean severity*	1.0	1.0	2.8	2.8
females	incidence	3/5	5/5	5/5	5/5
	mean severity*	1.0	1.2	2.8	3.4

(* severity not specified by the authors)

The marginally higher incidence and/or severity of vacuolated hepatocytes in the low dose group was considered to be not treatment-related (can be recorded in untreated rats) in contrast to effects at the mid enad high dose.

Degenerative cardiomyopathy:

males		0 mg/kg	15 mg/kg	50 mg/kg	75 mg/kg
	incidence	3/5	1/5	3/5	5/5
	mean severity*	1.7	1.0	1.3	2.4
females	incidence	0/5	0/5	3/5	3/5
	mean severity*	-	-	1.3	1.3

(* severity not specified)

Treatment-related effects on the heart in females at > 50 mg/kg and in males at 75 mg/kg.

Organ weights (differences in organ weights (in % of controls; n = 5):

		15 mg/kg	50 mg/kg	75 mg/kg
Males	kidney absolute	-4	-1	+9
	kidney relative	-3	+7	+21
	liver absolute	0	-4	+22
	liver relative	+2	+5	+36
Females	kidney absolute	+7	+10	+16
	kidney relative	-2	+6	+17
	liver absolute	+6	+32	+36
	liver relative	-3	+28	+37

These effects in the liver of males and females correlated with hepatocellular hypertrophy and/or vacuolated hepatocytes.

Conclusions:

No toxic effects were recorded at 15 mg/kg in males and females; a slightly higher body weight gain and food consumption was seen in females (also at higher dose levels). In males at >= 50 mg/kg ptyalism, a lower body weight gain, minimal to marked vacuolated hepatocytes accompanied by lower cholesterol and triglyceride levels, paleness and accentuated lobular pattern of the liver were observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

19.22 mg/kg bw/day

Study duration:

subacute

Species:

rat

Organ:

heart

liver

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Read across: 2 -Mercaptoethanol

No studies regarding the repeated dose toxicity of sodium 2 -mercaptoethanolate were available. Therefore, data on the structural analogue 2 -mercaptoethanol (CAS: 60 -24 -2) has been used.

CIT (2003) reported a combined repeated dose toxicity study with a reproduction/developmental toxicity screening test (OECD guidelines 407 and 422). Male and female rats were given orally doses (gavage) of 15, 50 and 75 mg/kg bw/d over a study period of about 7 weeks. No toxic effects were recorded at 15 mg/kg bw/day in males and females. A slightly higher body weight gain and food consumption was seen in treated females (not significant, also at higher dose levels) whereas in males at ≥ 50 mg/kg bw/day a significant lower body weight gain (-11 to -24%) compared to controls was detected. Ptyalism was observed in both genders at the mid and high dose levels. Males showed the following effects on the liver: at ≥50 mg/kg bw/day paleness and accentuated lobular pattern of the liver at necropsy, histopathology revealed minimal to marked vacuolated hepatocytes accompanied by lower blood cholesterol (0, 50, 75 mg/kg bw/day: 1.6 vs. 0.9 and 0.6 mmol/L) and triglyceride levels (0.85 vs. 0.39 and 0.23 mmol/L); at 75 mg/kg/day, absolute and relative liver weight (22 and 36%, respectively) were significantly increased, as well as minimal to slight hypertrophy of hepatocytes. In females at the dose levels of 50 and 75 mg/kg bw/day absolute (32 and 36%, respectively) and relative liver weight (28 and 37% respectively) were significantly increased, and paleness of the liver was detected at termination; histopathological examinations showed minimal to slight liver cell hypertrophy, and increased incidence and severity of vacuolated hepatocytes. Incidence and severity of cardiomyopathy was increased in males at 75 mg/kg/day and in females at 50 and 75 mg/kg bw/day. Hematology, seminology and urine analysis did not show any alterations. The NOAEL for male and female rats was determined to be 15 mg/kg bw/day. After correction for the molecular weight the NOAEL was determined to be 19.22 mg/kg bw/day for sodium 2 -mercaptoethanolate.

Justification for classification or non-classification

Repeated oral administration of the read across substance 2-mercaptoethanol resulted in clinical symptoms (excessive salivation, decreased body weight in males) and effects on the liver (organ weight increased and vacuolated liver cells in both genders) and the heart (degenerative cardiomyopathy in females at ≥ 50 mg/kg bw/day, in males at 75 mg/kg bw/day). Thus, the read across substance 2-mercaptoethanol is classified for Specific Target Organ Toxicity (STOT RE cat.2; target organs: liver, heart) based on the effects observed at ≥ 50 mg/kg bw/day according to CLP (Regulation (EC) No. 1272/2008. The same classification (STOT RE 2) thus also applies for sodium 2 -mercaptoethanolate.