Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted:
- Ames test with S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 (met. act.: with and without) (OECD TG 471, GLP); tested up to cytotoxic concentrations
- Mammalian cell gene mutation assay with CHO cells (HPRT test) (met. act.: with and without) (OECD Guideline 476, pre-GLP); tested up to cytotoxic concentrations; read-across from DOPA-Glycinate
- In vitro mammalian chromosome aberration test with cultured human lymphocytes (met. act.: with and without) (OECD Guideline 473, pre-GLP); tested up to cytotoxic concentrations; read-across from DOPA-Glycinate

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
- they are manufactured from similar or identical precursors under similar conditions
- they share structural similarities with common functional groups (corresponding to scenario 2 of the read-across assessment framework): both, the target and source substance, are aliphatic amines with C8-18 alkyl chains and acetate functions
- Two thirds (w/w) of the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (excluding the solvent water) are composed of the source substance DOPA-Glycinate. The remaining third of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid consists of other aliphatic amines and derivatives which are considered as structural analogues to those constituting the source substance DOPA-Glycinate and may therefore be expected to elicit comparable (eco)toxicological effects.

The read-across hypothesis is based on structural similarity of target and source substances. Based on available experimental data, including key physicochemical properties and data from acute toxicity, repeated dose toxicity, genotoxicity and short term ecotoxicity studies, the read-across hypothesis is supported by a quite similar toxicological profile of both substances.

(Eco)toxicological, physicochemical and environmental fate data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

Therefore, read-across from the existing ecotoxicity, environmental fate and toxicity studies conducted with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Further details are attached to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For further details refer to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
For further details refer to IUCLID section 13.

4. DATA MATRIX
For further details refer to IUCLID section 13.

Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
- they are manufactured from similar or identical precursors under similar conditions
- they share structural similarities with common functional groups (corresponding to scenario 2 of the read-across assessment framework): both, the target and source substance, are aliphatic amines with C8-18 alkyl chains and acetate functions
- Two thirds (w/w) of the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (excluding the solvent water) are composed of the source substance DOPA-Glycinate. The remaining third of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid consists of other aliphatic amines and derivatives which are considered as structural analogues to those constituting the source substance DOPA-Glycinate and may therefore be expected to elicit comparable (eco)toxicological effects.

The read-across hypothesis is based on structural similarity of target and source substances. Based on available experimental data, including key physicochemical properties and data from acute toxicity, repeated dose toxicity, genotoxicity and short term ecotoxicity studies, the read-across hypothesis is supported by a quite similar toxicological profile of both substances.

(Eco)toxicological, physicochemical and environmental fate data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

Therefore, read-across from the existing ecotoxicity, environmental fate and toxicity studies conducted with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Further details are attached to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For further details refer to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
For further details refer to IUCLID section 13.

4. DATA MATRIX
For further details refer to IUCLID section 13.

Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Type of assay:
mammalian cell gene mutation assay
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Remarks:
The use of E.coli WP2 or S. typhimurium TA102 was not mandatory at that time.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
+ TA1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 0.12, 0.37, 1.11, 3.33, 6.67 and 10.0 mg/mL as test material; in a pretest for toxicity the test substance was very toxic for TA 98 at a dose level of 5.0 mg per plate and above, both in the absence and in the presence of the S-9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 d

NUMBER OF REPLICATIONS: 3 + independent repeat

DETERMINATION OF CYTOTOXICITY
- Method: background lawn
Evaluation criteria:
A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate
observed with the vehicle, together with evidence of a dose-response.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

From the results obtained in both experiments it appeared that incubation of the test substance with the bacteria did not increase the number of his+ revertants with S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, either in the absence or in the presence of the S-9 mix.

At dose levels of 667.7 and 1000.0 µg per plate the test substance was toxic for all strains both in the absence and in the presence of the S-9 mix, as was seen from a reduced background lawn of bacterial growth. With strains TA 1535, TA 1537, TA 1538 and TA 98 slight toxicity was also observed at a dose level of 3.33 µg per plate.

The positive controls used in the present assays gave the expected strong increase in the number of his+ revertants, both in the absence and in the presence of the S-9 mix.

Conclusions:
Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 either in the absence or in the presence of the S-9 mix, under the conditions employed in this evaluation.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1983), Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (27% aqueous solution) in water in concentrations of 0 (control), 0.12, 0.37, 1.11, 3.33, 6.67 and 10.0 mg/mL (in terms of test material) in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method.

The test substance was tested up to cytotoxic concentrations.

In a pretest for toxicity the test substance was very toxic for TA 98 at a dose level of 5.0 mg per plate and above, both in the absence and in the presence of the S-9 mix. With strains TA 1535, TA 1537, TA 1538 and TA 98 slight toxicity was also observed at a dose level of 3.33 µg per plate.

The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the tester strains. Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 under the conditions employed. 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
+ TA1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316 and 1000 μg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 per concentration and experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants by at least 50%, clearing or diminution of the background lawn or degree of survival of the treated cultures
Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- Biological relevance of the results should be considered first.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Preliminary study:

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 1000 μg/plate in both experiments. Hence, 1000 μg test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study:

Six concentrations ranging from 3.16 to 1000 μg/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 316 and 1000 μg/plate in all test strains. In addition, cytotoxicity (reduction of the number of revertants by more than 50%) was noted in both experiments without metabolic activation at the concentration of 100 μg/plate in test strain TA1535. The reduction of the number of revertants by more than 50% in test strain TA1537 in the plate incorporation test with metabolic activation at 3.16 μg/plate is considered to be caused by the high variation in individual counts and not due to cytotoxicity above all as no concentration response relationship was noted.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to cytotoxic concentrations in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

Conclusions:
Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid did not show mutagenic activity in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 either in the absence or in the presence of the S-9 mix, under the conditions employed in this evaluation.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (26.6% aqueous solution) in water in concentrations of 0 (control), 3.16, 10.0, 31.6, 100, 316 and 1000 μg per plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method; the confirmatory assay was performed as preincubation assay.

The test substance was tested up to cytotoxic concentrations. The substance was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 1000 μg/plate in both experiments. Hence, 1000 μg test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

In the main test, pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 316 and 1000 μg AMPHOLYT 51/plate in all test strains. In addition, cytotoxicity (reduction of the number of revertants by more than 50%) was noted in both experiments without metabolic activation at the concentration of 100 μg/plate in test strain TA1535. The reduction of the number of revertants by more than 50% in test strain TA1537 in the plate incorporation test with metabolic activation at 3.16 μg/plate is considered to be caused by the high variation in individual counts and not due to cytotoxicity above all as no concentration response relationship was noted.

The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the tester strains. Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 under the conditions employed. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A reverse gene mutation assay in bacteria according to OECD guideline 471 is available for the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid. A mammalian cell gene mutation assay (HPRT test), and an in vitro mammalian chromosome aberration test are available for the structurally closely related source substance DOPA-Glycinate. A justification for read-across is attached to IUCLID section 13.

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (26.6% aqueous solution) in water in concentrations of 0 (control),3.16, 10.0, 31.6, 100, 316 and 1000 μg per plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method; the confirmatory assay was performed as preincubation assay.

The test substance was tested up to cytotoxic concentrations.The substance was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 1000 μg/plate in both experiments. Hence, 1000 μg test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

In the main test, pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 316 and 1000 μg test item/plate in all test strains. In addition, cytotoxicity (reduction of the number of revertants by more than 50%) was noted in both experiments without metabolic activation at the concentration of 100 μg/plate in test strain TA1535. The reduction of the number of revertants by more than 50% in test strain TA1537 in the plate incorporation test with metabolic activation at 3.16 μg/plate is considered to be caused by the high variation in individual counts and not due to cytotoxicity above all as no concentration response relationship was noted.

The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the tester strains. Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strainsTA 98, TA 100, TA 102, TA 1535 and TA 1537under the conditions employed. 

The mutagenic potential of DOPA-Glycinate was tested in the bacterial reverse mutation test using the plate incorporation assay. The study was conducted according to OECD guideline 471 (1997). The substance was tested up to cytotoxic concentrations.No increase in the number of revertant colonies was found in any of the tested strains with or without metabolic activation while the positive controls gave the expected increase in the mean number of revertant colonies.Under the conditions of this study, the test item dissolved in water was not mutagenic.

The clastogenic potential of DOPA-Glycinate was tested in human lymphocytes. The study was carried out according to OECD guideline 473 (1983). The substance was tested up to cytotoxic concentrations. The test item did not induce structural chromosome damage in cultured human lymphocytes either in the presence or in the absence of S9-mix.

The in vitro genotoxicity of DOPA-Glycinate was tested in Chinese hamster ovary cells (HPRT assay). The study was carried out according to OECD guideline 476 (1984). The substance was tested up to cytotoxic concentrations. In the absence and in the presence of a metabolic activation system, the test item did not induce a significant increase in the mutant frequency in both independent assays.

Based on the overall negative results of in vitro genotoxicity testing, teh target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid may be regarded as void of any genotoxic potential.

No human data are available for genetic toxicity. However, there is no reason to believe that the negative results would not be relevant to humans.





Justification for classification or non-classification

Based on the available data, the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 with respect to mutagenicity.