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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
One in vitro genetic toxicity assay (Ames Test) conducted with SDIBP and two in vitro (In vitro Mammalian Chromosome Aberration Test and Mammalian Cell Gene Mutation Test) and one in vivo (in vivo bone marrow erythrocyte micronucleus) genetic toxicity studies from the members of hydrotropes, i.e. sodium xylene sulphonate (CAS No. 1300-72-7) and sodium cumene sulphonate ( CAS 28348-53-0), were assessed. All studies are guideline studies, conducted according to GLP requirements and fully documented. The conclusion from validated assays is the test substance is not mutagenic, and not genotoxic.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
June to July 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented study according to OECD test guideline
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Test material information:
Composition 1
Species:
mouse
Strain:
other: BOR:NMRI (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: young adults
- Weight at study initiation: 30.4 +/- 1.7 g (males); 24.3 +/- 1.5 g (females)
- Assigned to test groups randomly: no data
- Fasting period before study: no
- Housing: 5 animals per cage, gender separated, macrolone cages Type III
- Diet : Ssniff R10 exclusive diet for rats (ad libitum); supplied by Ssniff Spezialfutter GmbH, Soest, Germany
- Water: drinking water (ad libitum)
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: July 9, 1991 To: July 18, 1991
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 40 %
- Amount of vehicle: 16.8 ml/kg bw
Frequency of treatment:
single oral application
Post exposure period:
24, 48 and 72 hours
Remarks:
Doses / Concentrations:
4467 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral by gavage
- Dose: 100 mg/kg bw
- Vehicle: water
- Total application volume: 10 ml/kg bw
- post exposure period: 24 hours
Tissues and cell types examined:
bone marrow; polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum tolerable dose (MTD)* was selected as dose.

The MTD was determined in a dose range finding study :
Phase 1: Limit test with 5000 mg/kg bw
Phase 2: Determination of the TMTD range with reduced animal number
Phase 3: Determination of the MTD with 5 animals/sex/dose

*MTD is defined as dose with no mortality but clear clinical symptoms within 3 days after application

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24, 48 and 72 hours after treatment the animals were killed by cervical dislocation and tissue was sampled.

DETAILS OF SLIDE PREPARATION:
The femora were removed and the bone marrow was suspended in fetal calf serum. The cell suspensions were centrifuged with 160 x g for 5 minutes and the supernatand discarded. The serum was resuspended and the suspension purified using a cellulose chromatographic column. The eluate was centrifuged at 800 x g for 10 minutes and the pellet in fetal calf serum /25 mM EDTA suspended. From this suspension 3-4 smears per animals were prepared on slides which were dried for at least 24 hours and stained with May-Grünwald/Giemsa solution.

METHOD OF ANALYSIS:
The cell analysis was performed by means of a Zeiss miscroscope at a 1000fold magnification (oil immersion). At least 1000 polychromatic erythrocytes (PCE) per animal were examined to determine the frequency of micronucleated cells. The ratio of PCE to normochromatic cells (NCO) was determined for a sample of 1000 erythrocytes. The number of micronucleated cells in counted NCE was determined.
Evaluation criteria:
For the identification of micronuclei the following criteria were considered:
a) roundish and clear contour by the nuclear membrane
b) diameter of about 1/20 of the size of the polychromatic erythrocyte
c) lays in the same focus layer as the observed erythrocyte

The micronucleus test is regarded as positive (test substance induces micronuclei in polychromatic erythrocytes) if the frequency of mucronucleated polychromatic erythrocytes of at least one tretament group is statistically significantly increased compared to the negative control and the increase is biologically relevant.

Statistics:
Mean values and standard deviations were calculated for the following parameters:
a) number of polychromatic erythrocytes (PCE) containing micronuclei
b) ratio of PCE/NCE

Comparison of treatment groups with different post exposure periods with negative controls of respective post exposure periods. After control of the relative frequency of micronuclei in the treatment groups on homogeneity with the mean relative frequency a statistical analysis of micronucleus frequency using a 2 x 2 contingency table with chi² test and continuity table according to Yates was performed (see [1]).
The differences of miconucleus frequencies in the positive control were reassessed in the two-sided t-test. This test was also used for the statistical analysis of the PCE/NCE-ratio.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
Phase 1: males and females: 5000 mg/kg bw (limit test);
Phase 2: males: 1585, 1995, 2512, 3162 and 3981 mg/kg bw; females: 3162, 3548, 3981, 4467 and 5000 mg/kg bw.
Phase 3: males and females: 3981, 4467 and 5000 mg/kg bw.

- Clinical signs of toxicity in test animals:
Phase 1: 4/5 male and 1/5 female animals died. The males died within 4 hours after application, the female died within 24 hours after application of the test substance. Clinical signs as hunched posture, ruffled fur, closed eyes and diarrhea were observed.
Phase 2: 1/5 female at 3981 mg/kg bw died. At high doses clinicals signs as hunched posture, ruffled fur, closed eyes and diarrhea were observed.
Phase 3: 2/5 male and 2/5 female animals died at 500 mg/kg bw. At 4467 mg/kg bw no mortality occured but severe toxic effects.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
Hunched posture, ruffled fur, closed eyes and diarrhea were observed. One day after application the effects subsided and two days after application all animals were free of clinical symptoms. One male and one female animal died 24 and 5.5 hours after application, respectively. These animals were replaced with mice of a concurrent satellite group also treated with the test substance. One male of the satellite group died 5 hours after application.

- Induction of micronuclei:
Positive control: Significantly increased number of polychromatic erythrocytes (PCE) with miconuclei both in male and female animals.
Test substance: No significant increase in the number of PCE with micronuclei after 24 and 48 hours in both males and females and after 72 hours in males. After 72 hours a statistically significant increase of the number of micronuclei in PCEs in females were observed. If the results of males and females were combined no significant difference in the frequency of micronuclei between treatment group and combined vehicle control group is observed. (Tables 1, 2 and 3)

- Ratio of PCE/NCE: Neither in the positive control group nor in the treated males a significant difference of PCE/NCE ratio compared to the control group is observed. In the females of the treatment groups no difference was observed 24 and 48 hours after treatment. After 72 hours the PCE/NCE ratio was decreased compared to vehicle control group but still was above the typically observed ratio of 1.0. (Tabels 1, 2 and 3)
- Appropriateness of dose levels and route: The selected dose level investigated as the maximum tolerable dose (MTD) induced clear toxic symptoms and was lethal in two animals. According to the guideline the recommended criteria for the dose level were fulfilled. The route of administration was appropriate as systemic availability could be demonstrated by clinical signs.

The statistically significant increase in frequency of micronuclei is considered to be of no biological relevance for the following reasons:

- The frequency of micronuclei at this sampling time point (0.18%) is not above the frequency of micronuclei of controls generally observed in this test laboratory (0.07 - 0.22%). The statistical significance in this case is caused by the low frequency of micronuclei in the control group (0.02%) which deviates downwards from the so far observed frequencies for controls. After addition of male and female animals of sampling time point 72 h into one group there is no more a statistically significant difference.

- A delayed effect based on slow excretion is improbable for sodium cumenesulphonate as sulphonic acids in general are readily absorbed and do not show any tendency for accumulation. But this kind of detention is regarded as prerequisite to explain based on the kinetic of erythrocyte maturation an impact on micronuclei frequency at sampling time point of 72 hours.

The results of the positive control affirm the sensitivity of the mouse strain to mutagenic substances.The frequency of micronuclei in polychromatic erythrocytes was considerably increased compared to the control group.

Table 1: Results of in vivo micronucleus test for male animals (mean ± standard deviation)

      Neg. control   test substance 4467 mg/kg bw      Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
micronuclei in 1000 PCE  0.8  ± 0.8 1.8  ± 0.8   0.4 ± 0.5 0.6 ± 0.5    0.8  ± 0,8   0.2  ± 0.4 48.0* ± 19.6
 % PCE with micronuclei 0.08  0.18  0.04 0.06  0.08 0.02  4.8
PCE / NCE  1.0 ± 0.2 1.4 ± 0.3  1.6 ± 0.3  1.0 ± 0.3 1.1 ± 0.4   1.9 ± 0.9 0.8  ± 0.1

*p < 0.05

Table 2: Results of in vivo micronucleus test for female animal (mean ± standard deviation)

      Neg. control   test substance 4467 mg/kg bw      Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
micronuclei in 1000 PCE 1.4  ± 1.7 1.8  ± 1.1   0.2 ± 0.4 2.2 ± 1.1    1.2  ± 0,8   1.8*  ± 1.5 36.8* ± 9.8
 % PCE with micronuclei 0.14  0.18  0.02 0.22  0.12 0.18 3.6
PCE / NCE  1.2 ± 0.2 1.4 ± 0.2  2.4 ± 0.6  1.0 ± 0.2 1.3 ± 0.3   1.3 ± 0.5 1.0  ± 0.1

*p< 0.05

Table 3: Results of in vivo micronucleus test for male + female animals (mean ± standard deviation)

      Neg. control   test substance 4467 mg/kg bw      Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
micronuclei in 1000 PCE  1.1  ± 1.3 1.8  ± 0.9  0.3 ± 0.5 1.4 ± 1.2    1.0  ± 0.8   1.0  ± 1.3 42.4* ± 15.7
 % PCE with micronuclei 0.11  0.18  0.03 0.14  0.10 0.10  4.24
PCE / NCE  1.1 ± 0.2 1.4 ± 0.3  2.0 ± 0.6  1.0 ± 0.2 1.2 ± 0.4   1.6 ± 0.8 0.9  ± 0.1

* p < 0.05

Conclusions:
Interpretation of results (migrated information): negative
All male mice treated with the test substance showed no statistically significant increase in micronucleus frequency at any sampling time compared to control animals. For the female mice treated with the test substance at sampling times 24 and 48 hours after treatment also no statistically significant increase in micronucleus frequency was observed. Only at sampling time point 72 hours a statistically significant increase of polychromatic erythrocytes with micronuclei compared to control animals was observed. This effect was regarded as biologically not relevant as this increase ís based on the exceptional low micronucleus frequency of vehicle control group.
Sodium cumenesulphonate under these test conditions is regarded as not mutagenic in the micronucleus test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

One bacterial cell gene mutation study (Ames Test) with four strains of Salmonella (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100),and E. coli WP2 uvr A, with and without S9 metabolic activation, positive and negative controls, and 5 exposure concentrations of test substance using both the Ames plate incorporation and pre-incubation methods, was performed with SDIBP (Huntsman, 2012). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Hence, SDIPB was considered to be non-mutagenic under the condition of this test.

The read-across was performed from sodium xylene sulphonate (CAS No. 1300-72-2) for in vitro and in vivo genetic toxicity study. Chinese hamster ovary mammalian chromosome aberration and mouse lymphoma gene mutation assays were carried out to sodium xylene sulphonate. The protocols and results of both assays reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA NIH, 1998. The mammalian cell chromosome aberration study in CHO with and without Aroclor 1254 metabolic activation, was tested up to limit concentrations (2513, 3750 and 5000 mg a.i./mL. The test substance, with and without metabolic activation was considered not genotoxic in this assay.

The mammalian cell gene mutation study using mouse lymphoma cells was performed by using five exposure concentrations of the test substance, with and without S9 metabolic activation (USA NIH, 1998). The genotoxicity results were negative without activation and equivocal with activation (one replicate was positive and the 2nd replicate was negative). The study conclusion was "negative for genotoxicity".

Anin vivobone marrow erythrocyte micronucleus test according to OECD guideline 474 was performed with sodium cumenesulphonate (CAS 28348-53-0) (Fedtke, 1992). All male mice treated with the test substance showed no statistically significant increase in micronucleus frequency at any sampling time compared to control animals. For the female mice treated with the test substance at sampling times 24 and 48 hours after treatment also no statistically significant increase in micronucleus frequency was observed. Only at sampling time point 72 hours a statistically significant increase of polychromatic erythrocytes with micronuclei compared to control animals was observed. This effect was regarded as biologically not relevant as this increase is based on the exceptional low micronucleus frequency of vehicle control group. Sodium cumenesulphonate under these test conditions is regarded as not mutagenic in the micronucleus test.

 

Category Justification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances.

Applying the category approach read-across concept to SDIBP, data will be used from a representative member of the hydrotropes category to avoid unnecessary (animal) testing. The endpoints for which the read-across approach to SDIBP is applied, are: toxicokinetics, short-term toxicity to fish, toxicity to microorganism, acute toxicity (inhalation and dermal), skin irritation/corrosivity, skin sensitisation, genetic toxicity (in vitro, in vivo), repeated-dose toxicity (oral and dermal), screening for carcinogenicity, and screening for reproductive / developmental toxicity.

Hazard assessment related key information for SDIBP:

SDIPB and the hydrotrope members exhibit similar levels of low toxicity. Acute toxicity values are above the classification limits, they are not sensitizing and show no genotoxic effects. Both exhibit neglible irritation to the skin, but are moderately irritating to the eyes (Cat 2B). Together with the chemical structure similarity and their similar chemical properties, it is deemed correct to fill the existing data gaps on mammalian toxicity of SDIPB with the data of the hydrotrope category.

Justification for classification or non-classification

According to the classification criteria, the SDIPB does not need to be classified for genotoxicity.