Registration Dossier

Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test compound: Thioglycolic acid
CAS no.: 68-11-1
Source: Arkema
Batch: 05.04.05.C
Purity: 80.22% (w/w) in water

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
- Animals
. Preliminary toxicity test: 12 male and 15 female mice were used,
. Main cytogenetic test: 55 mice, 27 males and 28 females were used,
. Blood sampling: six mice, three males and three females were used.
. Strain: Swiss Ico: OF1 (IOPS Caw).
. Breeder: Charles River Laboratories, l'Arbresle, France.
. Age: on the day of treatment, the animals were approximately 6 weeks  old.
. Acclimation: at least 5 days before the day of treatment.

- Environmental conditions
· Temperature: 22 ± 2°C,
· Relative humidity: 30 to 70%,
· Light/dark cycle: 12 h/12 h (07:00 - 19:00),
· Ventilation: at least 12 cycles/hour of filtered non-recycled fresh air.
. Housing: individually in polycarbonate cages (24.0 x 13.5 x 13.0 cm)  containing autoclaved sawdust (SICSA, Alfortville, France).

- Food and water
. Food: A04 C pelleted maintenance diet (SAFE, Villemoisson-sur-Orge,  France), ad libitum.
. Water: drinking water filtered by a FG Millipore membrane (0.22  micron), ad libitum.

Administration / exposure

Route of administration:
dermal
Vehicle:
- Formulation of the test substance:
-Vehicle: Purified water (CIT, Millipore) degassed by sonication for at  least 15 minutes and saturation with nitrogen gas for at least 15 min . 
-Treatment volume: 3 mL/kg.
-Formulation procedure: All the dose-levels and concentrations were  expressed as active item, taking into account the active material content  of 80.22% (w/w).
-For the preliminary tests: Treatment with neutralized test item: the maximum achieved  concentration of the preparation neutralized  using NaOH (10 M) was 497  mg/mL (obtained pH ranging from 6.92 to 7.24). Using a treatment volume  of 3 mL/kg, the target dose-level was 1491 mg/kg/day.
-Remaining treatments: The test item preparations were brought to pH  ranging from 3.75 to 4.21, by addition of NaOH (10 M). The preparations  were used at the concentrations of 666.6, 500, 333.3, 250 and 166.67  mg/mL, in order to reach the target dose-levels of 2000, 1500, 1000, 750  and 500 mg/kg/day, respectively, using a treatment volume of 3 mL/kg.  All the test item dosage forms were prepared extemporaneously. In  addition, except for the preparation at 666.6 mg/mL (target dose-level of  2000 mg/kg/day), the preparations were made under nitrogen atmosphere and  were stored at room temperature and under nitrogen atmosphere until  treatment.
-For the main cytogenetic test: The test item dosage forms were  prepared extemporaneously under nitrogen atmosphere and were stored at  room temperature and under nitrogen atmosphere until treatment. The pH of the preparations was adjusted to a value of approximately 4  (ranging from 4.0 to 4.10) by addition of NaOH (10 M).  The preparations were used at the concentrations of 41.67, 83.33, 166.67  and 333.3 mg/mL, in order to reach the target dose-levels of 125, 250,  500 and 1000 mg/kg/day, respectively, using a treatment volume of 3  mL/kg. 
Details on exposure:
- Preparation of the animals:
On the day before the beginning of the treatment period, an area of  approximately 1,5 x 2 cm or 2,5 x 2 cm (3 to 5 cm2), estimated to be up  to 10% of the total body surface (1) was clipped free from hair, as close  to the skin as possible on the dorsum of the animals, with an electric  clipper. Before the first dosage form application, the treatment area was examined  and any animals showing skin abnormality and/or irritation was replaced  from the spare animals ordered.  During the treatment period, the animals were clipped whenever necessary,  at least 4 hours before dosing.

- Administration
The test item formulated in a vehicle was applied in a film as thin and  uniform as possible, to the clipped area of the skin. The dosage forms  was applied to the dorsum using a micropipette.  No dressing or protective plastic collar was used. Each animal was given the dosage forms once a day, on two consecutive  days, at approximately the same time.

- Preliminary toxicity test
In order to determine the highest dose-level, several preliminary tests  were performed on groups of six animals (three males and/or three  females). Clinical signs, any local reaction at the treatment site  (following the below-mentioned scoring scale) and any mortality was  recorded over a period of 48 hours. At the end of this period, the  animals were killed by CO2 inhalation in excess.
Duration of treatment / exposure:
2 administrations at 24-h interval
Frequency of treatment:
daily
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
Males: 1000, 500 and 250 mg/kg. Females: 500, 250 and 125 mg/kg
Basis:

No. of animals per sex per dose:
5 in the control groups and in the low and intermediate dose groups. 7 or 8 in the high dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA), batch No. 4A121 (Endoxan, Baxter, Maurepas,  France) dissolved in distilled water at a concentration of 5 mg/mL.
-route: oral
-treatment volume: 10 mL/kg
-number of administrations: one

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
- Preparation of the bone marrow smears:
At the time of sacrifice, all the animals were killed by CO2 inhalation  in excess. The femurs of the animals were removed and the bone marrow was  flushed out using fetal calf serum. After centrifugation, the supernatant  was removed and the cells in the sediment were resuspended by shaking. A  drop of this cell suspension was placed and spread on a slide. The slides  were air dried and stained with Giemsa. The slides were coded so that the  scorer is unaware of the treatment group of the slide under evaluation  ("blind" scoring).

- Microscopic examination of the slides:
For each animal, the number of the micronucleated polychromatic  erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the  polychromatic (PE) and normochromatic (NE) erythrocyte ratio was  established by scoring a total of 1000 erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, a statistically significant  increase in the frequency of MPE must be demonstrated when compared to  the concurrent vehicle control group. Reference to historical data, or  other considerations of biological relevance was also taken into account  in the evaluation of data obtained.
Statistics:
Normality and homogeneity of variances was tested using a Kolmogorov  Smirnov test and a Bartlett test. If normality and homogeneity of variances are demonstrated, the  statistical comparisons were performed using a Student t-test (2 groups)  or a one-way analysis of variance (>= 3 groups) followed by a Dunnett  test (if necessary). If normality or homogeneity of variances are not demonstrated, a  Mann/Withney test (2 groups) or a Kruskall Wallis test (>= 3 groups) was  performed followed by a Dunn test (if necessary). All these analyses were performed using the software SAS Enterprise Guide  V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05  for all tests.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

- PRELIMINARY TOXICITY TEST
. Treatment with neutralized test item (target pH of 7):
At 1491 mg/kg/day, no mortality and no clinical signs were noted in the  three males and three females treated with the test item. In one male  (1/3), a very slight erythema was noted since 24h after the first  treatment until sacrifice.
. Treatment with partially neutralized test item (target pH of 4):
For all dose-levels tested no cutaneous reactions were noted in any of  the treated animals.
At 2000 mg/kg/day, 2/3 males and 2/3 females were found dead 16 hours  following the first treatment. The two surviving animals (one male and  one female) were sacrificed and no second treatment was performed.
At 1500 mg/kg/day (males only), 2/3 males were found dead 17 hours  following the first treatment. The surviving animal was sacrificed and no  second treatment was performed.
At 1000 mg/kg/day, no deaths occurred in males, either hypoactivity,  half-closed eyes and piloerection or only piloerection were observed  following the second treatment. In females, one animal (1/3) was found  dead more than 16 hours following the second treatment. The two surviving  females showed hypoactivity, lateral recumbency, tremors, piloerection  and/or half-closed eyes.
At 750 mg/kg/day (females only), one animal (1/3) was found dead 21 hours  following the first treatment. The two surviving females showed poor  clinical conditions and were therefore sacrificed immediately.
At 500 mg/kg/day (females only), no deaths occurred. Dyspnea and  piloerection were noted in 3/3 females following the second treatment. 

- CYTOGENETIC TEST
In males treated at 1000 mg/kg/day, mortality was observed (3/7).  Piloerection was noted in the surviving animals.
In males treated at 500 mg/kg/day, piloerection was noted (3/5).
No clinical signs and deaths were observed in males treated at 250  mg/kg/day as well as in females during the study.
For all dose-levels tested no cutaneous reactions were noted in any of  the treated animals.
For both males and females, the mean values of MPE as well as the PE/NE  ratio in the groups treated with the test item, were equivalent to those  of the vehicle group.
Cyclophosphamide induced a significant increase in the frequency of MPE,  indicating the sensitivity of the test system under our experimental  conditions. The study was therefore considered valid.

Results of the cytogenetic test: data summary

Group

Doses(1) (mg/kg/day)

MPE/1000PE

PE/NE ratio

Time of sacrifice after the last administration

mean

(sd)

mean

(sd)

Males

Vehicle

 -

2.1

(0.5)

0.5

(0.2)

24 h

Test item

250

2.3

(1.7)

0.6

(0.1)

500

1.6

(1.1)

0.6

(0.1)

1000

1.1

(0.5)

0.6

(0.2)

Cyclophosphamide

50

19.6

(4.2)*

0.5

(0.0)

Females

Vehicle

-

0.9

(0.5)

0.6

(0.1)

24 h

Test item

125

1.7

(0.8)

0.8

(0.2)

250

0.9

(0.7)

0.9

(0.4)

500

1.5

(0.7)

0.8

(0.3)

Cyclophosphamide

50

15.1

(6.2)**

0.7

(0.1)

(1)expressed as active item

Five animals per group (except for the high-dose treated group of males: four animals)

MPE: Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes

NE: Normochromatic Erythrocytes

sd: standard deviation

Statistical significance:* p < 0.05 ** p < 0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
THIOGLYCOLIC ACID did not induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two cutaneous administrations, at a 24 hour interval, at the dose-levels of 250, 500 and 1000 mg/kg/day for males or 125, 250 and 500 mg/kg/day for females.
Executive summary:

The clastogenic potential of thioglycolic acid was evaluated in a micronucleus assay on mouse bone marrow performed according to the OECD guideline # 474. Thioglycolic acid, partially neutralized to pH 4, was administered dermally over a 2-day period to three groups of five male and five female Swiss mice at dose-levels of 0, 250, 500 and 1000 mg/kg bw/day for males or 0, 125, 250 and 500 mg/kg/day for females. The positive control was the cyclophosphamide administered orally. The polychromatic erythrocytes/normochromatic erythrocytes ratios (PE/NE) in the treated groups were equivalent to those of the control groups. However, systemic exposure was confirmed by the mortality observed in males given 1000 mg/kg/day thioglycolic acid, as well as by the clinical signs observed in males treated with 1000 and 500 mg/kg/day thioglycolic acid. No increase of the frequency of the micronucleated polychromatic erythrocytes was observed in the bone marrow harvested 24 hours after the last treatment. Positive and vehicle controls gave the expected results.