Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation assay

The potential of diethylhydroxylamine to induce reverse mutation inSalmonella typhimuriumwas evaluated in an OECD TG #471 study (Haddouk, 2001a). Diethylhydroxylamine was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteriaSalmonella typhimurium:TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to 312.5, 625, 1250, 2500 and 5000 µg/plate of diethylhydroxylamine in distilled water (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. No toxicity was noted towards all the strains used, both with and without S9 mix. Diethylhydroxylamine did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five tester strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. In conclusion, diethylhydroxylamine did not show mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

The potential of diethylhydroxylamine to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells was evaluated in an OECD TG # 476 assay (Haddouk, 2001b). After a preliminary toxicity test, diethylhydroxylamine was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5 x 106cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Cytotoxicity was then determined using cloning efficiency (CE2) before expression of the mutant phenotype. Cell viability (using cloning efficiency CE2) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Other parameters such as cell concentration, relative survival (RS), relative suspension growth (RSG) and/or relative total growth (RTG) were also taken into consideration. The selected treatment-levels were156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment, and625, 1250, 2500, 3750 and 5000 µg/mL for the second experiment. The cloning efficiencies CEo and CE2and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Without S9 mix, a slight to moderate toxicity was induced, depending on the dose-levels. In both experiments, a noteworthy and dose-related increase in the mutation frequency, accompanied with an increase in the number of small colonies, was noted at dose-levels 1250 µg/mL. With S9 mix, a slight to moderate toxicity was induced, mainly at the highest dose-level tested. On the basis of the positive criteria specified in the study plan, no positive response could be attributed to the test item. However it should be noted that an increase in the number of small colonies was generally observed in both experiments. In conclusion, diethylhydroxylamine induced mutagenic activity in the mouse lymphoma assay, without S9 mix, possibly from a clastogenic origin.

Chromosomal aberration assay

In an in vitro genotoxicity assays, diethylhydroxylamine appeared to be clastogenic.

The potential of diethylhydroxylamine to induce chromosome aberrations in cultured human lymphocytes was evaluated in an OECD TG #473 study (Haddouk, 2002). Diethylhydroxylamine was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) and incubated at 37°C, for 48 hours. In the first experiment, lymphocyte cultures were exposed to concentrations of 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL diethylhydroxylamine or control items, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. In the second experiment, cells were exposed without S9 mix to concentrations of 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL diethylhydroxylamine or control items either for 3 hours and then rinsed or continuously until harvest, or with S9 mix, for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. Without S9 mix, a moderate to strong decrease in the mitotic index was noted, depending on the dose-level and the treatment duration. A significant and reproducible increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-hour treatment at dose-levels>2500 µg/mL. After the 44-hour treatment, a slight and significant increase in the frequency of cells with structural chromosomal aberrations was noted at 312.5 µg/mL. With S9 mix, a slight to strong decrease in the mitotic index was noted, depending on the dose-level and the harvest time. No increase in the frequency of cells with structural chromosomal aberrations which could be considered as relevant was noted, at both harvest times. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. In conclusion, diethylhydroxylamine induced chromosome aberrations in cultured human lymphocytes, without metabolic activation (S9 mix).

Link to relevant study records

Referenceopen allclose all

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:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (from Sprague-Dawley rats, treated by  Aroclor 1254)
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9-mix . sodium azide (TA1535 and TA100, 1µg/plate), 2-nitrofluorene (TA98, 0.5 µg/plate), mitomycin C (TA102, 0.5 µg/plate), 9-aminoacridine (TA 1537, 50µg/plate). With S9-mix: 2-anthramine (all strains, 2 µg/plate except 10 µg/plate for TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) excepted for the 2nd assay with S9 (preincubation)

DURATION
-  Pre-incubation time: 60 minutes
- Pre-incubation temperature: 37°C
- Incubation time: 48 to 72 hours
- Incubation temperature: 37°C

NUMBER OF REPLICATIONS:  3/dose

EXAMINATION:
- Bacterial toxicity (performed on TA98, TA100 and TA102 at 0, 10, 100,  500, 1000, 2500 and 5000 µg/plate)
- Number of revertants / plate. 
Two identical assays were performed.
Evaluation criteria:
- Positivity criteria for genotoxicity:
. number of revertants in the vehicle control is consistent with  laboratory historical data,
. number of revertants in the positive control is higher than that of  vehicle control and is consistent with laboratory historical data,
. number of revertants at least twice that of negative control revertants,
. reproducibility of the positive response.
Statistics:
Not appropriate
Species / strain:
S. typhimurium, other: TA 1535, TA1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
All the dose-levels are expressed as active substance, taking into  account the purity of 86.733%.

BACTERIAL TOXICITY
No toxicity was noted towards the three strains used, with and without  S9-mix.

GENOTOXIC EFFECTS
The number of revertants for the vehicle and positive controls was as  specified in the acceptance criteria. The study was therefore considered  valid.  Since the test substance was freely soluble and non-toxic, the highest  dose-level was 5000 µg/plate, according to the criteria specified in the  international guidelines.  The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000  µg/plate, for both mutagenicity experiments with and without S9 mix.  In the TA 1535 strain in the first experiment without S9 mix, a 2-fold  increase in the number of revertants was noted at 1250 µg/plate. This  increase was not reproducible (not observed in the second experiment  performed under the same experimental conditions). In addition the  revertant values obtained remained clearly within the vehicle historical  range (3-23). Therefore, this slight increase in revertants was not  considered as relevant. 

PRECIPITATION
No precipitate was observed in the Petri plates

TEST-SPECIFIC CONFOUNDING FACTORS
none
Conclusions:
DIETHYLHYDROXYLAMINE does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The potential of diethylhydroxylamine to induce reverse mutation in Salmonella typhimurium was evaluated in an OECD TG #471 study.Diethylhydroxylamine was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to 312.5, 625, 1250, 2500 and 5000 µg/plate of diethylhydroxylamine in distilled water (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. No toxicity was noted towards all the strains used, both with and without S9 mix. Diethylhydroxylamine did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five tester strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. In conclusion, diethylhydroxylamine did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
primary culture, other: Human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S-9 prepared by rat liver enzyme induction  by Aroclor 1254
Test concentrations with justification for top dose:
78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/ml for the first experiment,
312.5, 625, 1250, 2500, 3750 and 5000 µg/ml for the second experiment.
(All the dose levels were expressed as active item, taking into account the purity)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: solubility
The test item was dissolved in the vehicle at the concentration of 275 mg/ml for both experiments.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C (3 µg/ml), Cyclophosphamide (25 and 50  µg/ml)
Details on test system and experimental conditions:
CELL CULTURE:
- Number of donors: 2
- Sex of donor: 1 man + 1 woman
- Age of donor: no data
- Cell cycle length: 12-14 hours
- Number of passages: none
- Method of maintenance of cell cultures: 0.5 mL of heparinised whole  blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf  serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100  l1g/rnL) and phytohaemagglutinin. The cultures were then placed at 37°C  for 48 hours.
- Metaphase arrest
·        Spindle poison used: colcemid (10 µg/ml)
·        Duration of exposure: 1.5 h
·        Time before harvest: 1.5 h
- Absence of mycoplasma: no data

TREATMENT
- Number of replicates: 2
- Metabolic activation:  S9 + Glucose-6-phosphate, NADP, KCl, MgCl2  and sodium phosphate buffer.
- Vehicle: culture medium
- Volume of vehicle added: 5 ml
- Cell density at seeding: no data
- Negative controls: vehicle 
- Pre-incubation time: 48h
- Incubation temperature: 37°C
- Incubation time: 20h and 44h
- Duration of exposure: 3h or 44h

DESCRIPTION OF FOLLOW-UP REPEAT STUDY: no data

EXAMINATION:
- Harvesting and fixation: cells are harvested in a KCl (0.075 M)  suspension and fixed by dropping in an ice-cold methanol/glacial acetic  acid (3:1 v/v) mix
- Slide preparation: cells are stained in Giemsa.
- Cytotoxicity test: Cytotoxicity was evaluated using the mitotic index,  which indicates whether a item induces mitotic inhibition.
- Number of metaphases analyzed: 200 / dose (only 50 metaphase/culture  were analysed when at least 10% cells with structural chromosome  aberrations were observed).
- Types of sought aberrations: gaps, chromatid and chromosome breaks and  exchanges, and others (multiple aberrations and pulverizations). In  addition, the following numerical aberrations were recorded when  encountered: polyploidy and endoreduplication.
- Microscope : the analysis of the slides was performed at Microptic,  cytogenetic services (2 Langland Close Mumbles, Swansea SA4 4 LY, UK) in  compliance with GLP
Evaluation criteria:
Acceptance criteria:
. the frequency of cells with structural aberrations in the vehicle  control was consistent with historical data
. the frequency of cells with structural chromosome aberrations in the  positive control was significantly higher than that of the control and  consistent with historical data.

Evaluation criteria:
A reproducible and statistically significant increase in the frequency of  cells with structural chromosome aberrations for at least one of the  dose-levels and one of the two harvest times was considered as a positive  result. Reference to historical data was also taken into account in the  evaluation of the findings.
Statistics:
When necessary, a Chi-square test was used  (with p=0.05)
Species / strain:
primary culture, other: Human lymphocytes
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=2500 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
primary culture, other: Human lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/ml (44-hour harvest time)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
VALIDATION CRITERIA:
The frequencies of cells with structural chromosome aberrations of the  vehicle and positive controls were as specified in acceptance criteria.  The study was therefore considered valid. 

CYTOTOXICITY: 
Without S9 mix, a moderate decrease in the mitotic index (up to 53%  decrease) was noted after the 3-hour treatment, mainly at dose-levels >  2500 µg/mL.  After the 44-hour treatment, a moderate to strong toxicity was induced at  the dose-levels tested (42-100% decrease in the mitotic index). 
With S9 mix, a slight to moderate decrease in the mitotic index (up to 45%  decrease) was noted at the 20-hour harvest time.  At the 44-hour harvest time, a marked to strong decrease in the mitotic  index was noted at dose-levels > 3750 µg/mL (60-87% decrease). 

CHROMOSOMAL ABERRATION ANALYSIS: 
Without S9 mix, a significant and reproducible increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-hour treatment at dose-levels > 2500 µg/mL (the frequency of aberrant  cells reached 6.5% or 10%, p < 0.001, in the first and second experiments  respectively). After the 44-hour treatment, a slight and significant  increase in the frequency of cells with structural chromosomal  aberrations was noted at 312.5 µg/mL (4%, p < 0.05). 
With S9 mix, in the first experiment, no significant increase in the  frequency of cells with structural chromosomal aberrations was noted,  whereas in the second experiment, a slight but a statistically significant (5%, p < 0.01) increase in the frequency of cells with  structural chromosomal aberrations was noted at 3750 µg/mL.  Since this increase was neither reproducible nor noted at higher  dose-levels, it was not considered as relevant.  No significant increase in the frequency of cells with structural  chromosomal aberrations was noted at the 44-hour harvest time. 

PRECIPITATION: no TEST-SPECIFIC 

CONFOUNDING FACTORS: none
Conclusions:
DIETHYLHYDROXYLAMINE induced chromosome aberrations in cultured human lymphocytes, without metabolic activation (S9 mix).
Executive summary:

The potential of diethylhydroxylamine to induce chromosome aberrations in cultured human lymphocytes was evaluated in an OECD TG #473 study. Diethylhydroxylamine was tested in two independent experiments, both withandwithout a liver metabolising system (S9 mix), obtainedfrom rats previously treated with Aroclor 1254. For each culture, heparinised whole blood was added to culture medium containing a mitogen(phytohaemagglutinin)and incubated at 37°C, for 48 hours. In the first experiment, lymphocyte cultures were exposed to concentrations of 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL diethylhydroxylamine or control items, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. In the second experiment, cells were exposed without S9 mix to concentrations of 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL diethylhydroxylamine or control items either for 3 hoursandthen rinsed or continuously until harvest, or with S9 mix, for 3 hoursandthen rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. Without S9 mix, a moderate to strong decrease in the mitotic index was noted, depending on the dose-levelandthe treatment duration. A significant and reproducible increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-hour treatment at dose-levels>2500 µg/mL. After the 44-hour treatment, a slight and significant increase in the of frequency of cells with structural chromosomal aberrations was noted at 312.5 µg/mL. With S9 mix, a slight to strong decrease in the mitotic index was noted, depending on the dose-levelandthe harvest time. No increase in thefrequency of cells with structural chromosomal aberrations which could be considered as relevant was noted, at both harvest times. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

In conclusion, diethylhydroxylamine induced chromosome aberrations in cultured human lymphocytes, without metabolic activation (S9 mix).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 obtained from the liver of rats treated  with Aroclor 1254
Test concentrations with justification for top dose:
without S9: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
with S9: 625, 1250, 2500, 3750 and 5000 µg/mL
(All the dose levels were expressed as active substance, taking into account the purity)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: solubility
The test item was dissolved in the vehicle at a concentration of 250 mg/ml for the preliminary toxicity test and both mutagenic experiments.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: methylmethanesulfonate (MMS,25 µg/ml). With metabolic activation: cyclophosphamide (CPA, 3 µg/ml).
Details on test system and experimental conditions:
CELL CULTURES
- Source: Biovalley (77601, Marne-la-Vallée, France)
- Culture type: suspension
- Number of passages: 1
- Method of maintenance of cell cultures: Before treatment, the cells  were seeded in 50 mL of RPMI 1640 medium containing 10% horse serum,  L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and  sodium pyruvate (200 µg/mL). The cells were then incubated at 37°C in a  humidified atmosphere of 5% CO2/95% air. After incubation, cells were  counted and culture medium was removed. Cells were suspended in adequate  volume of RPMI 1640 medium containing 10% inactivated horse serum. 
- Cell density: 500.000 cells/ml
- Deficiences/Proficiences: TK
- Absence of mycoplasma: no data

TREATMENT
- Number of replicates: 2
- Volume of vehicle added: 50 ml
- Pre-incubation time: (If applicable)
- Incubation temperature: 37°C
- Duration of exposure: 3h
- Selective agent: TFT (4 µg/ml)
- Duration of expression period: 11-12 days

EXAMINATION
- Cytotoxicity: 1.6 cells/well (one 96-well plate/culture = two  plates/dose-level, except for the vehicle control where two 96-well  plates/culture were used = total of four plates) to determine  cytotoxicity using cloning efficiency after treatment (CEo). After at  least 7 days of incubation at 37°C, the clones were counted. RCEo  (survival relative to vehicle control after treatment) was then  calculated. CEo = -ln (empty wells/total wells) / number of cells per well RCEo = CEo treated / CEo  vehicle control x 100
- Cell viability: 1.6 cells/well (one 96-well plate/culture = two  plates/dose-level, except for the vehicle control where two 96-well  plates/culture were used = total of four plates) to define the number of  viable cells (CE2 = Cloning Efficiency  at the end of the expression  period). After at least 7 days of incubation at 37°C, the clones were  counted. RCEo (viability relative to vehicle control after treatment) was  then calculated.
- Mutations assessment: 2000 cells/well (one 96-well plate/culture = two  plates/dose-level, except for the vehicle control where two 96-well  plates/culture were used = total of four plates) to select the  TFT-resistant mutant cells (for determination of CEmutant). After 11-12  days of incubation at 37°C in the presence of 4 µg TFT/ml of culture  medium, the clones were counted, differentiating small and large colonies. The relative mutant frequency is calculated as MF = CEmutant / CE2 x 10E6

NUMBER OF INDEPENDENT EXPERIMENTS: 2

PRELIMINARY TEST:
To assess cytotoxicity, cells were exposed to 0, 10, 100, 500, 1000, 2500  and 5000 µg/ml, with and without S9-mix. CEo and RCEo were determined.
Evaluation criteria:
Difference between small and large colonies was based on the following  criterion:
. size of small colonies: <25% of the diameter of the well
. size of large colonies: >25%  of the diameter of the well.
For scoring of colonies in mutant plates, the following parameters were  considered:
. well with mutant colony (small or large)
. well without mutant colony.
When small and large colonies were present in the same well, two mutant  colonies were counted (one small + one large).

Acceptance criteria:
-        the cloning efficiency of the vehicle controls were between 0.6 and 1.4  for CEo and between 0.7 and 1.3 for CE2.
-        the mutation frequency of the vehicle control was between 60 and 250 x  10E6
-        the mutation frequency of positive control was higher than that of the  vehicle control and consistent with historical data.

Evaluation criteria: 
a  reproducible two-fold increase in the mutant  frequency when compared to the vehicle control, at any dose-level and/or  evidence of a dose relationship were considered as positive results.
Statistics:
None
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
VALIDITY CRITERIA:
The cloning efficiencies CEo and CE2 and the mutation frequencies of the  vehicle and positive controls were as specified in acceptance criteria.  The study was therefore considered valid.

PRELIMINARY TEST: 
Except for some slight decreases in the cloning efficiency immediately after treatment (CEo) or in the relative survival (RS), no noteworthy  toxicity was induced at the dose-levels tested, both with and without S9  mix.

CYTOTOXICITY: 
Without S9 mix, a slight to moderate toxicity was induced as shown by  31-61 % decrease in the CEo, 28-60% decrease in the relative total growth  (RTG) as well as 35-70% decrease in the RS. 
With S9 mix, a slight to moderate toxicity was induced, mainly at the  highest dose-level tested, as shown by: up to 60% decrease in the CEo and  the RS as well as 44-48% decrease in the RTG. 

MUTAGENICITY: 
Without S9 mix, in both experiments, a noteworthy and dose-related  increase in the mutation frequency (up to 3.4 fold the vehicle control  value), accompanied with an increase in the number of small colonies, was  noted at dose-levels > 1250 µg/mL.
With S9 mix, on the basis of the positive criteria specified in the study  plan, no positive response could be attributed to the test item. However  it should be noted that an increase in the number of small colonies was  generally observed in both experiments.
Conclusions:
DIETHYLHYDROXYLAMINE induced mutagenic activity in the mouse lymphoma assay, without S9 mix.
Executive summary:

The potential of diethylhydroxylamine to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells was evaluated in an OECD TG # 476 assay. After a preliminary toxicity test, diethylhydroxylamine was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.Approximately 0.5 x 106cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Cytotoxicity was then determined using cloning efficiency (CE2) before expression of the mutant phenotype. Cell viability (using cloning efficiency CE2) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Other parameters such as cell concentration, relative survival (RS), relative suspension growth (RSG) and/or relative total growth (RTG) were also taken into consideration. The selected treatment-levels were156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment, and 625, 1250, 2500, 3750 and 5000 µg/mL for the second experiment. The cloning efficiencies CEo and CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Without S9 mix, a slight to moderate toxicity was induced, depending on the dose-levels. In both experiments, a noteworthy and dose-related increase in the mutation frequency, accompanied with an increase in the number of small colonies, was noted at dose-levels 1250 µg/mL. With S9 mix, a slight to moderate toxicity was induced, mainly at the highest dose-level tested. On the basis of the positive criteria specified in the study plan, no positive response could be attributed to the test item. However it should be noted that an increase in the number of small colonies was generally observed in both experiments. In conclusion, diethylhydroxylamine induced mutagenic activity in the mouse lymphoma assay, without S9 mix.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
performed only whitout S9
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
thioguanine resistant mutations
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
obtained from Argonne National Laboratory;
Metabolic activation:
without
Test concentrations with justification for top dose:
39-195 mM
Vehicle / solvent:
Culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
Confluent 100-mm diameter plates of V-79 cells, grown in Dulbecco's Modified Eagles Medium (DME) containing 10% fetal calf serum (FCS), 0.5 mM glutamine, and 100 units/ml of penicillin and 100 µg/ml streptomycin, were treated with a series of concentrations of the test compounds in medium for three hours, washed with Dulbecco's phosphate buffet saline (PBS) solution and detached using 0.5% trypsin-EDTA solution. The cells were plated onto five 100-mm diameter plates at 2 x 10e5 cells/plate. After 3-4 days of expression the cells were treated with trypsin and reseeded at 2 x 10e5 cells/plate onto five new 100-mm diameter plates. Three to four days later the cells were replated at 8 x 10e4 cells/100-mm diameter plate for mutation measurement and at 100 cells/plate for determination of plating efficiency. Twenty-four hours lacer, 200 µl of 0.5 mg/ml thioguanine was added to the mutation plates. After 7-10 days of incubation in 5% C02 at 37°C, the colonies were stained with Giemsa stain, and large, visible (diameter >1 mm) colonies were counted.

NUMBER OF REPLICATIONS: 4
Evaluation criteria:
No data
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
EC50 for inhibition of DNA synthesis = 13 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Diethylhydroxylamine did not show thioguanine-resistant mutations in V-79 Chinese hamster cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Further in vivo genotoxicity assays were performed to explore the clastogenic activity observed in the in vitro assays. No clastogenic activity was observed in the polychromatic erythrocytes of the bone marrow of mice dosed orally with dose levels inducing systemic toxicity and a decrease of the ratio of polychromatic erythrocytes to total erythrocytes. As well, no UDS activity was observed in the hepatocytes from rats dosed orally with dose levels inducing clinical signs of toxicity.

The potential of diethylhydroxylamine to induce chromosome aberrations in mice was evaluated in an OECD TG #474 study (Putman and Gudi, 1995). Male and female ICR mice were dosed with diethylhydroxylamine by a single gavage administration at dose levels of 375, 750 or 1500 mg/kg. The volume administered was 20 ml/kg body weight. The high dose was estimated to be 80% of the LD50/3. Distilled water was used as the vehicle. Mortality was observed in 4/20 male and 5/20 female mice receiving 1500 mg/kg. Clinical signs following dose administration included lethargy and tremors in male mice and lethargy in female mice at 1500 mg/kg. Slight reductions (no greater than 21 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed relative to the respective vehicle controls in male and female mice, predominantly at the mid and high dose levels. No significant increases in micronucleated polychromatic erythrocytes were observed relative to their respective vehicle controls in male or female ICR mice at 24, 48 or 72 hours after dose administration (p > 0.05, Kastenbaum-Bowman). The results of the assay indicate that under the conditions described in this report, Diethylhydroxylamine did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female ICR mice. Diethylhydroxylamine was concluded to be negative in the mouse micronucleus assay.

Diethylhydroxylamine was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats using anin vivo/in vitroprocedure according to the OECD TG #486 (Beevers, 2003). Groups of four male rats were treated once with the vehicle (purified water), Diethylhydroxylamine (at 800 mg/kg or 2000 mg/kg) or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment). Clinical signs observed included piloerection and lethargy (2-4 hour experiment, 2000 mg/kg dose group). In the 12-14 hour experiment slight weight loss was observed (2000 mg/kg dose group). Approximately 2-4 hours (Experiment 1) or 12-14 hours (Experiment 2) after dosing, animals were sacrificed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, each animal and dose group. Negative (vehicle) control animals gave a group mean NNG value of less than 0.2 with only 0 to 1% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to 2.9 or more with more than 20% cells found to be in repair. Treatment with 800 or 2000 mg/kg Diethylhydroxylamine did not produce a group mean NNG value greater than zero nor were any cells found in repair at either dose. It was concluded that Diethylhydroxylamine did not induce UDS detectable under the experimental conditions employed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK.
- Age at study initiation: 8 weeks
- Weight at study initiation: 198-238 g (males)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups of no more than four animals of the same sex, in solid-floored cages,
cleaned
- Diet (ad libitum): Special Diets Services Ltd, RM1.(E).SQC.
- Water (ad libitum): bottled water (public
supply)
- Acclimation period: 5/6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 52-91
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
purified water
Details on exposure:
- dose volume: 10 mL/kg.
Frequency of treatment:
single administration
Post exposure period:
2-4 hour or 12-14 hour
Remarks:
Doses / Concentrations:
2000 and 800 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Positive control(s):
2-Acetamidofluorene (2-AAF) for the 12-14 hour experiment and dimethylnitrosamine (DMN) for the 2-4 hour experiment.
Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Available information indicated that the LD50 value for  Diethylhydroxylamine was greater than 2000 mg/kg (the maximum recommended  dose for the UDS assay). Accordingly, a confirmatory range-finder study  was performed at 2000 mg/kg. Additionally, both male and female animals  were tested in the range-finder study to determine any substantial  inter-sex differences in toxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
In the main study groups of four male rats were treated once with the  vehicle (purified water), Diethylhydroxylamine (at 800 mg/kg or 2000  mg/kg) or the required positive control, by oral gavage, at a dose volume  of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene  (2-AAF) suspended in corn oil (12-14 hour experiment) and 10 mg/kg  dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour  experiment).

DETAILS OF SLIDE PREPARATION:
- Killing of animals and preparation of hepatocyte cultures
For Experiments 1 and 2, animals were sacrificed a nominal 2-4 hours and 12-14 hours after dosing, respectively.
Individual animals were anaesthetized with Halothane and maintained under deep anaesthesia to prevent any likelihood of recovery. The liver was surgically exposed, the hepatic portal vein and superior vena cava cannulated with suitable cannulars and the liver perfused with suitable buffers. Approximately 400 mL of calcium free Buffer 1 was pumped at a flow rate of approximately 40 mL/min to wash the liver free of blood. The liver was then perfused with Buffer 2 also at a flow rate of 40 mL/min for approximately 5 minutes. Both buffers were gassed with 5% CO2 in air (v/v) prior to use and Buffer 2 throughout perfusion.
Calcium and collagenase (approximately 50 mg of collagenase dissolved in 1 mL of 769 mM CaCl2 and 10 mL of Buffer 2) was added to the reservoir and after one to two minutes the waste line was placed in the Buffer 2 reservoir so that the perfusate recirculates and the flow rate was reduced to 20 mL/min. When the reticular pattern of the liver had begun to break up and the liver became 'spongy', the perfusion was stopped. The liver was cut free into a suitable container with Buffer 2. The liver was transferred to a sterile dish, cut open and the hepatocytes carefully teased out. The resulting hepatocyte suspension was gently washed through 150 mm nylon mesh with Williams E medium-Complete (WE-C) to a volume of approximately 100 mL. Of this suspension, approximately 50 mL was taken and centrifuged at approximately 40 x 'g' for two to three minutes. The resultant pellet was resuspended in WE-C. The centrifugation and resuspension procedure was repeated at least twice and the pellet resuspended finally in approximately 20 mL WE-C. A sample (0.5 mL) of this suspension was taken, diluted with an equal volume of 0.4% (w/v) trypan blue in phosphate buffered saline (PBS) and the proportion of viable cells (those with unstained nuclei) determined using an haemocytometer. The culture was diluted where possible to provide approximately 1.5 x 105 viable cells/mL. Three mL of hepatocyte suspension was added to each well of a six-well multiplate containing 25 mm round plastic coverslips and incubated at 37°C ± 1°C in a 5% CO2 in air (v/v) atmosphere for at least 90 minutes to allow cells to attach.
- Radiolabelling of hepatocyte cultures
Medium was removed from the cells and the monolayers washed with 2 mL Williams E medium-Incomplete (WE-I) which was then replaced with 2 mL WE-I containing 10 mCi/mL [3H] thymidine. After 4 hours incubation at 37°C in a 5% CO2 in air (v/v) atmosphere, the medium was removed and the cells washed with three changes of WE-I containing 0.25 mM thymidine. Cultures were then incubated overnight with 3 mL of the same medium.
To prepare for autoradiography, coverslips were washed with 2 mL phosphate buffered saline (PBS) and the cells fixed with three changes of 2 mL glacial acetic acid:ethanol (1:3 v/v). The coverslips were then washed four times with purified water, allowed to dry and mounted onto previously labelled microscope slides, cells side up, with DPX.
- Autoradiography
Three of the six slides from each animal were coated in Ilford K2 liquid emulsion using a dipping technique. Each slide was dipped individually into the molten emulsion, ensuring that no air-bubbles were generated. After gelling over ice, for 10 minutes face upwards, the slides were incubated in a light-tight box at room temperature for approximately 90 minutes to let the emulsion dry. The slides were then packed in light-tight boxes containing desiccant, sealed with tape and refrigerated for 14 days. At the end of this time, the emulsion was developed in Kodak D19 developer and fixed using Ilford Hypam fixer. The cell nuclei and cytoplasm were then stained with Meyers haemalum/eosin Y. Slides were then dehydrated in ethanol, cleared in xylene and mounted with coverslips for microscopic examination. The spare, duplicate sets of slides were not required.
- Grain counting
Grain counting was performed using a microscope with a video camera connected to an image analysis system (Perceptive Instruments) and a computer programmed for automatic data capture.
Each slide was examined to ensure that the culture was viable. A patch of cells was selected as a starting point and cells were scored in a regular fashion by bringing new cells into the field of view, moving only in one axis. If the desired number of cells had not been scored before coming to the edge of the slide, the stage was moved one or two fields on the other axis and counting resumed. The circular field was centred over the nucleus of a suitable cell and the grains counted. The field of view was moved and counts obtained for three separate adjacent areas of cytoplasm. Nuclear and mean cytoplasmic grain counts were then recorded, and the net grains/nucleus (NNG) determined. 100 cells were analysed per animal, where possible using two of the three slides in each case.
Evaluation criteria:
The following criteria were used for cell analysis:
1. only cells with normal morphology were scored
2. isolated nuclei with no surrounding cytoplasm were not scored
3. cells without nuclear and/or cytoplasmic graining were not scored
4. cells with unusual staining artefacts were not scored
5. heavily labelled cells in S-phase were not scored
6. all other normal cells, 100 per animal were scored
7. all slides were analysed blind (coded).

Treatment of data
The following were calculated for each slide, animal and dose point:
1. the population average NNG and standard deviation (SD)
2. the percent of cells responding or in repair (ie ³ 5 NNG)
3. the population average cytoplasmic and nuclear grain count.

Acceptance criteria
The study would be considered valid if the negative control animals had a group mean NNG value that did not exceed the upper limit of the historical range. The positive control treatments should have group mean values of five or more NNG with 50% or more cells having NNG counts of five or greater.
Evaluation criteria
The test article would be considered as positive in this assay if, at any dose and at either time point:
1. the test article yielded group mean NNG values greater than 0 NNG and 20% or more of cells responding (mean NNG values >= 5)
2. an increase above solvent control levels was seen in both NNG and the percentage of cells in repair.
Cytoplasmic and nuclear grain count values as well as the concurrent negative control data would be considered in relation to the overall NNG values of cultures from treated animals.
If the test article failed to induce UDS at any dose tested after both 2-4 and 12-14 hours exposure, it would be considered clearly negative in this system.
Statistics:
None
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the range-finder study, groups of three male and three female out bred  Han Wistar (Crl:WI (Glx/BRL/Han) BR) rats were dosed once with up to 2000  mg/kg Diethylhydroxylamine. During a 2 day post-dose observation period  male animals dosed at 2000 mg/kg showed clinical signs of eye closure,  piloerection, lethargy and weight loss. Female animals dosed at 2000  mg/kg showed clinical signs of eye closure, piloerection, lethargy,  abnormal gait and being cold to the touch. However, due to the severity  of these signs the female animals were killed in extremis. Therefore, a  further three female animals were dosed at 1400 mg/kg and acceptable  clinical signs including piloerection, lethargy, abnormal gait, being  cold to the touch and weight loss were observed. Accordingly, as no  substantial inter-sex differences in toxicity were observed, male animals  only were tested in the main study at a maximum dose of 2000 mg/kg. A  lower dose level of 800 mg/kg (40% of the maximum dose) was also selected.

RESULTS OF DEFINITIVE STUDY
Clinical signs observed in the main study included piloerection and  lethargy (2-4 hour experiment, 2000 mg/kg dose group). In the 12-14 hour  experiment slight weight loss was observed (2000 mg/kg dose group). Approximately 2-4 hours (Experiment 1) or 12-14 hours (Experiment 2)  after dosing, animals were sacrificed and their livers perfused with  collagenase to provide a primary culture of hepatocytes. Cultures were  made from three animals in each dose group and were treated with [3H]  thymidine. Six slides from each animal were prepared with fixed  hepatocytes and of these, three were dipped in photographic emulsion to  prepare autoradiograms. Slides were examined microscopically after  development of the emulsion and staining, and the net grain count (NNG),  the number of grains present in the nucleus minus the mean number of  grains in three equivalent areas of cytoplasm, was determined for each of  two of the three slides, each animal and dose group. Negative (vehicle) control animals gave a group mean NNG value of less  than 0.2 with only 0 to 1% cells in repair. Group mean NNG values were  increased by 2-AAF and DMN treatment to 2.9 or more with more than 20%  cells found to be in repair. It may be noted that the Experiment 1  vehicle control NNG value was slightly higher than normal (0.2), however  this value is within the negative historical control range and therefore  acceptable. Additionally, although the positive control response for DMN  was lower than normal (falling outside of historical range), the response  was considered to be clearly indicative of UDS. Accordingly, in this  study the vehicle control NNG value was consistent with both published  and historical control data, and the system was shown to be sensitive to  two known DNA damaging agents requiring metabolism for their action. The  assay was therefore accepted as valid.  Treatment with 800 or 2000 mg/kg Diethylhydroxylamine did not produce a  group mean NNG value greater than zero nor were any cells found in repair  at either dose.
Conclusions:
When treated once via oral gavage with Diethylhydroxylamine at doses up to 2000 mg/kg male Han Wistar rats showed no induction of UDS in hepatocytes isolated ex vivo approximately 2-4 or 12-14 hours after dosing. It is concluded that Diethylhydroxylamine had no genotoxic activity detectable in this test system under the experimental conditions employed.
Executive summary:

Diethylhydroxylamine was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats using anin vivo/in vitroprocedure. Information available indicated that the LD50value for Diethylhydroxylamine was greater than 2000 mg/kg (the maximum recommended dose for the UDS assay). Accordingly, a confirmatory range-finder study was performed at 2000 mg/kg. Additionally, both male and female animals were tested in the range-finder study to determine any substantial inter-sex differences in toxicity. In the range-finder study, groups of three male and three female out bred Han Wistar (Crl:WI (Glx/BRL/Han) BR) rats were dosed once with up to 2000 mg/kg Diethylhydroxylamine. During a 2 day post-dose observation period male animals dosed at 2000 mg/kg showed clinical signs of eye closure, piloerection, lethargy and weight loss. Female animals dosed at 2000 mg/kg showed clinical signs of eye closure, piloerection, lethargy, abnormal gait and being cold to the touch. However, due to the severity of these signs the female animals were killedin extremis. Therefore, a further three female animals were dosed at 1400 mg/kg and acceptable clinical signs including piloerection, lethargy, abnormal gait, being cold to the touch and weight loss were observed. Accordingly, as no substantial inter-sex differences in toxicity were observed, male animals only were tested in the main study at a maximum dose of 2000 mg/kg. A lower dose level of 800 mg/kg (40% of the maximum dose) was also selected.

In the main study groups of four male rats were treated once with the vehicle (purified water), Diethylhydroxylamine (at 800 mg/kg or 2000 mg/kg) or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment). Clinical signs observed in the main study included piloerection and lethargy (2-4 hour experiment, 2000 mg/kg dose group). In the 12-14 hour experiment slight weight loss was observed (2000 mg/kg dose group). Approximately 2-4 hours (Experiment 1) or 12-14 hours (Experiment 2) after dosing, animals were sacrificed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, each animal and dose group.

Negative (vehicle) control animals gave a group mean NNG value of less than 0.2 with only 0 to 1% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to 2.9 or more with more than 20% cells found to be in repair. It may be noted that the Experiment 1 vehicle control NNG value was slightly higher than normal (0.2), however this value is within the negative historical control range and therefore acceptable. Additionally, although the positive control response for DMN was lower than normal (falling outside of historical range), the response was considered to be clearly indicative of UDS. Accordingly, in this study the vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid. Treatment with 800 or 2000 mg/kg Diethylhydroxylamine did not produce a group mean NNG value greater than zero nor were any cells found in repair at either dose.

It was concluded that Diethylhydroxylamine did not induce UDS detectable under the experimental conditions employed.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks
- Body weight at study initiation: 24.0 to 32.8 g (males) and 24.6 to  30.6 g (females)
- Assigned to test groups randomly: yes, computer-generated program based on body weight
- Fasting period before study: not specified
- Housing: up to 5 per plastic cages
- Diet (ad libitum): certified laboratory rodent chow
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 74 +/- 6
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
Distilled water
Details on exposure:
Administration volume: 20 ml/kg
Frequency of treatment:
Single administration
Post exposure period:
24, 48 or 72hours
Remarks:
Doses / Concentrations:
0, 375, 750 and 1500 mg/kg
Basis:

No. of animals per sex per dose:
5 males + 5 females (an additional group of 5 males and 5 females was designated as  replacement animals in the event of mortality prior to the scheduled  sacrifice and was dosed with the test article high-dose level.)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide 40 mg/kg
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
- Clinical observations (after dose administration and daily thereafter)
- Tissue examined: bone marrow
- Slide preparation:  At the scheduled sacrifice times, five mice per sex per treatment were  sacrificed by CO2 asphyxiation. Immediately following sacrifice, the  femurs were exposed, cut just above the knee, and the bone marrow was  aspirated into a syringe containing fetal bovine serum. The bone marrow  cells were transferred to a capped centrifuge tube containing  approximately 1 mL fetal bovine serum. The bone marrow cells were  pelleted by centrifugation at approximately 100 g for five minutes and  the supernatant was drawn off, leaving a small amount of serum with the  remaining cell pellet. The cells were resuspended by aspiration with a  capillary pipet and a small drop of bone marrow suspension was spread  onto a clean glass slide. Two to four slides were prepared from each  animal. The slides were fixed in methanol, stained with  May-Gruenwald-Giemsa and permanently mounted. 
- Number of cells analyzed per animal: 1000
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not  exceed 5/1000 polychromatic erythrocytes (0.5%) in the negative (vehicle)  control. The incidence of micronucleated polychromatic erythrocytes in the  positive control group must be significantly increased relative to the  negative control (p=<0.05, Kastenbaum-Bowman tables)
Statistics:
Statistical significance is determined with Kastenbaum-Bowman tables,  which is based on the binomial distribution.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Mortality at 1500 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
MORTALITY:  Mortality occurred within a day after dose administration as follows: 1/5 males at 1500 mg/kg 5/5 males and females at 3000 and 5000 mg/kg

CLINICAL SIGNS: Lethargy at 1500 mg/kg. One female exhibited tremors at 1500 mg/kg

NUMBER OF MICRONUCLEATED ERYTHROCYTES PER ANIMALS:  The number of micronucleated polychromatic erythrocytes was not  statistically increased relative to their respective vehicle control in  either males of females, regardless of dose level or bone marrow  collection time (cf. Attached Document)

PROPORTION OF IMMATURE ERYTHROCYTES AMONG TOTAL ERYTHROCYTES (PCE/NCE  RATIO):  Slight reductions (no greated than 21%) in some groups relative to controls
Conclusions:
The negative (vehicle) and positive controls fulfilled the requirements for determination of a valid test. Diethylhydroxylamine did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

Male and female ICR mice were dosed with Diethylhydroxylamine by a single gavage administration at dose levels of 375, 750 or 1500 mg/kg. The volume administered was 20 ml/kg body weight. The high dose was estimated to be 80% of the LD50. Distilled water was used as the vehicle. Mortality was observed in 4/20 male and 5/20female mice receiving 1500 mg/kg. Clinical signs following dose administration included lethargy and tremor in male mice and lethargy in female mice at 1500 mg/kg. Slight reductions (no greater than 21%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed relative to the respective vehicle controls in male and female mice, predominantly at the mid and high dose levels. No significant increases in micronucleated polychromatic erythrocytes were observed relative to their respect vehicle controls in male or female ICR mice at 24, 48 or 72 hours after dose administration (p>0.05, Kastenbaum-Bowman). The results of the assay indicated that diethylhydroxylamine did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or femaleICRmice. Diethylhydroxylamine was concluded to be negative in the mouse micronucleus assay.

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

Additional information

Several reliable genetic toxicity studies are available for diethylhydroxylamine. Diethylhydroxylamine is negative in a bacterial mutagenicity assay with and without metabolic activation, positive in the in vitro chromosome aberration study using human lymphocytes without metabolic activation, positive in the mouse lymphoma gene mutations assay without metabolic activation possibly from a clastogenic origin and negative in the HPRT gene mutations assay on V79 Chinese hamster cells without metabolic activation). Further in vivo genotoxicity assays were performed to explore the clastogenic activity observed in the in vitro assays. No clastogenic activity was observed in the polychromatic erythrocytes of the bone marrow of mice dosed orally with dose levels inducing systemic toxicity and a decrease of the ratio of polychromatic erythrocytes to total erythrocytes. As well, no UDS activity was observed in the hepatocytes from rats dosed orally with dose levels inducing clinical signs of toxicity.Therefore, diethylhydroxylamine is not considered as a genotoxic substance.

Justification for classification or non-classification

No classification is warranted for germ cell mutagenicity according to REGULATION (EC) No 1272-2008.