[2-(1-ethoxyethoxy)ethyl]benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria: Not mutagenic (read-across from Hyacinth body #3 tested in OECD TG 471 - Ames test)

- In vitro chromosomal aberrations (OECD TG 487): Not mutagenic

- Gene mutations in mammalian cells (OECD TG 490): Not mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Result derived from read across is sufficiently reliable because all Annex XI criteria are met.

Justification for type of information:

The read across justification is presented in the genetic toxicity endpoint summary and the accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results of the OECD TG 471 study 9Ames) for read-across substance Hyacinth body #3, Hyacinth body is not considered to be mutagenic in bacteria.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2000 - 26 May 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Hyacinth body.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix

Test concentrations with justification for top dose:

50 to 5000 μg per plate in the presence and absence of S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: according to guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate incorporation
DURATION: Exposure duration: 48h -72h
NUMBER OF REPLICATIONS: The experiment was performed in triplicate and repeated in full after an interval of at least 3 days.
DETERMINATION OF CYTOTOXICITY
- Method: Background lawn measurement and reduction in revertant colonies compared to the controls

Statistics:

Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was done, using a X2-test (Mohn and Ellenberger,1977).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): A historical overview of the revertant frequencies of the strains used in the Freiburger Labor fur Mutagenitätsprüfung for the years 1998 to 2000.
Mix: -S9 +S9 -S9 +S9
strain: spontaneous /solvent cont./ spontaneous/ solvent cont
TA1535: 31±11 (13/58) 30±11 (10/62) 19±6 (9/27) 18±6 (9/28)
TA1537: 11±4 (5/20) 11±4 (6/21) 16±5 (9/29) 16±5 (9/29)
TA98: 30±8 (18/47) 29±8 (19/50) 40±10 (21/63) 38±9 (23/62)
TA100: 150±35 (89/226) 138±29 (94/205) 139±38 (88/238) 133±38 (79/222)
TA102: 280±37 (218/359) 269±38 (163/337) 306±45 (237/400) 298±46 (207/404)

The historical data of positive controls are given in the form: strain, mutagen (concentration of mutagen in μg/plate), mean of revertants per plate (minimum/maximum). The data of test without S9 are: TA1535, NaN3 ( 0.7), 513±130 (242/688); TA1537, 9-AA (50), 278±97 (131/529); TA98, 2-NF (2.5), 353±113 (190/632); TA100, NaN3 ( 0.7), 427±90 (312/665); TA102, Mitmomycin C (0.15) 820±151 (577/1061). The data of the tests with lot KH3799 of S9 are: TA1535, 2-AA (1.5), 547±205 (310/1118); TA1537, 2-AA (1.5), 299±157 (100/543); TA98, 2-AA (0.7), 919±458 (319/2243); TA100, 2-AA (0.7), 854±336 (402/1955); TA102, 2-AA (1.5), 1116±337 (610/1799). Additional data of the tests with lot KH3799 of S9 are: TA100, B(a)P (5.0), 869±100 (736/976)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence and absence of S9-mix Resedafol / Corps 302 was bacteriotoxic towards the strains TA1535 at 1500 µg/plate and towards the strains TA98, TA100, TA102, and TA1537 at 5000 μg/plate.

The number of spontaneous revertants observed using each of the five strains was close to those previously established in the laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). The results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the fall activity of the metabolizing system.

Conclusions:

Under the conditions of this study according to OECD TG 471 (Ames test), Hyacinth body #3 was determined to be not mutagenic.

Executive summary:

The mutagenic activity of Hyacinth body #3 was evaluated in accordance with OECD 471 guideline (Ames test) and according to GLP principles, therefore a Klimisch 1 rating was assigned. The test was performed as a standard plate incorporation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the presence and absence of S9-mix, Hyacinth body #3 was toxic towards the strains TA1535 at 1500 µg/plate and towards the strains TA98, TA100, TA102, and TA1537 at 5000 μg/plate. No precipitation was observed at any of the concentrations. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 TA102), both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study, it is concluded that Hyacinth body #3 is not mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2016 - 02 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014. A new version of the guideline has been adopted by OECD. The study procedures described in this report are also in compliance with this new guideline:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no 487: In Vitro Mammalian Cell Micronucleus Test (adopted 29 July 2016).

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
Average Generation Time (AGT): 12.7 - 13.0 h
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3 hr exposure; 27 hr fixation: 52, 164, 512, 1600 and 1943 μg/mL
Without S9-mix, 24 hr exposure; 24 hr fixation: 52, 164, 512, 1600 and 1943 μg/mL

First cytogenetic test:
Without and with S9-mix, 3 hr exposure; 27 hr fixation: 100, 200, 300, 350, 400, 450 and 500 μg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 3 hr exposure; 27 hr fixation: 200, 350 and 400 μg/mL
With S9-mix, 3 hr exposure; 27 hr fixation: 200, 400 and 450 μg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 50, 70, 90, 110, 130, 150 and 170 μg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 90 and 150 μg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. Hyacinth body was dissolved in dimethyl sulfoxide of spectroscopic quality. DMSO is accepted and approved by authorities and international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine: 0.1 μg/mL for 3 h exposure period; 0.05 μg/mL for 24 h exposure period without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 46 ± 2 hr
- Exposure duration:
Short-term treatment: Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment: Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 μg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Histolab, Gothenburg, Sweden) and mounted with a coverslip in an automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

NUMBER OF CELLS EVALUATED: at least 1000 mononucleated cells/culture and at least 1000 binucleated cells/culture

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and of osmolality: The pH and osmolarity of a concentration of 512 μg/ml were 7.61 and 449 mOsm/kg respectively (compared to 7.68 and 455 mOsm/kg in the solvent control).
- Precipitation: At a concentration of 1600 μg/mL Hyacinth body precipitated in the culture medium. Further investigation showed that a concentration of 512 μg/mL already precipitated in the culture medium after 3 and 24 hours of incubation.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 512 μg/mL and above in the presence and absence of S9, 3 hr treatment/27 hr fixation and at dose levels of 164 μg/mL and above in the absence of S9 for the continuous treatment of 24 hr/24 hours fixation.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

Experiment 1:
Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time

Concentration (µg/ml) Cytostasis (%) Number of mononucleated cells with micronuclei 1) Number of binucleated cells with micronuclei 1)
1000 1000 2000 1000 1000 2000
A B A+B A B A+B
0 0 2 1 3 3 2 5
200 17 1 0 1 3 2 5
350 30 0 1 1 3 5 8
400 46 2 2 4 7 4 11
0.25 MMC-C 37 4 3 7 31 37 68***
0.1 Colch 88 23 32 55*** 4 2) 6 2) 10

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time

Concentration (µg/ml) Cytostasis (%) Number of mononucleated cells with micronuclei 1) Number of binucleated cells with micronuclei 1)
1000 1000 2000 1000 1000 2000
A B A+B A B A+B
0 0 0 0 0 4 3 7
200 13 0 1 1 2 3 5
400 36 0 1 1 4 3 7
450 58 2 0 2 6 5 11
15 CP 59 3 4 7* 36 19 55***
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2) 813 and 679 binucleated cells were scored for the presence of micronuclei, respectively.

Experiment 2:

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time

Concentration (µg/ml) Cytostasis (%) Number of mononucleated cells with micronuclei 1) Number of binucleated cells with micronuclei 1)
1000 1000 2000 1000 1000 2000
A B A+B A B A+B
0 0 0 1 1 6 8 14
10 5 0 0 0 1 4 5
90 22 0 0 0 3 8 11
150 38 3 0 3 1 5 6
0.15 MMC-C 46 1 0 1 9 21 30*
0.23 MMC-C 56 1 1 2 19 14 33**
0.05 Colch 95 21 24 45*** 6 2) 3 2) 9

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2) 417 and 330 binucleated cells were scored for the presence of micronuclei, respectively.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

Experiment 1:

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml Culture Number of cells with ….nuclei CBPI
1 2 3 or more
0 A 241 242 17 1.55
B 212 274 14 1.60
100 A 262 222 16 1.51
B 247 243 10 1.53
200 A 268 222 10 1.48
B 271 219 10 1.48
300 A 300 200 10 1.43
B 291 203 6 1.43
350 A 302 192 6 1.41
B 302 193 5 1.41
400 A 345 153 2 1.31
B 357 149 3 1.30
450 A 422 77 1 1.16
B 432 66 2 1.14
500 A 482 18 0 1.04
B 479 21 0 1.04
0.25 MMC-C A 306 194 0 1.39
B 332 169 0 1.34
0.38 MMC-C A 344 156 0 1.31
B 342 157 1 1.32
0.1 Colch A 473 25 2 1.06
B 469 24 7 1.08

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time

Concentration µg/ml Culture Number of cells with ….nuclei CBPI
1 2 3 or more
0 A 232 255 13 1.56
B 213 265 22 1.62
100 A 253 239 8 1.51
B 214 272 14 1.60
200 A 262 223 15 1.51
B 253 237 12 1.52
300 A 268 219 13 1.49
B 273 217 10 1.47
350 A 289 203 8 1.44
B 266 229 5 1.48
400 A 312 180 8 1.39
B 321 174 5 1.37
450 A 371 127 2 1.26
B 386 114 0 1.23
500 A 459 41 0 1.08
B 441 59 0 1.12
15 CP A 372 128 0 1.26
B 388 112 0 1.22
17.5 CP A 407 92 1 1.19
B 402 98 0 1.20

Experiment 2:

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time

Concentration µg/ml Culture Number of cells with ….nuclei CBPI
1 2 3 or more
0 A 128 304 68 1.88
B 123 316 61 1.88
10 A 146 304 50 1.81
B 126 315 59 1.87
50 A 152 308 40 1.78
B 142 327 31 1.78
70 A 161 309 31 1.74
B 133 334 33 1.80
90 A 191 286 23 1.66
B 186 277 37 1.70
110 A 191 295 14 1.65
B 196 293 11 1.63
130 A 230 265 5 1.55
B 213 277 10 1.59
150 1) A 234 264 2 1.54
B 228 266 6 1.56
170 1) A 293 203 4 1.42
B 281 214 5 1.45
0.15 MMC-C A 268 231 1 1.47
B 259 239 2 1.49
0.23 MMC-C A 307 191 2 1.39
B 317 178 5 1.38
0.05 Colch A 478 20 1 1.04
B 476 23 1 1.05

1) The test item precipitated in the culture medium

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mononucleated Binucleated
- S9-mix + S9-mix - S9-mix
3 hour exposure 24 hour exposure 3 hour exposure 3 hour exposure 24 hour exposure
Mean number of micronucleated cells
(per 1000 cells) 21.11 22.57 26.02 28.78 23.18
SD 28.25 27.75 12.96 25.69 15.59
n 210 204 108 210 204
Upper control limit
(95% control limits) 78.68 86.40 48.42 70.48 63.33
Lower control limit
(95% control limits) -36.45 -41.26 3.61 -12.92 -16.97

SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between January 2012 and June 2016.

- Negative (solvent/vehicle) historical control data:
Mononucleated Binucleated
+ S9-mix - S9-mix + S9-mix - S9-mix
3 hour exposure 3 hour exposure 24 hour exposure 3 hour exposure 3 hour exposure 24 hour exposure
Mean number of micronucleated cells
(per 1000 cells) 0.89 1.07 0.95 3.57 3.77 4.00
SD 0.92 1.10 1.27 2.55 2.48 2.62
n 102 104 99 102 104 99
Upper control limit
(95% control limits) 3.04 3.87 3.84 9.19 10.23 10.81
Lower control limit
(95% control limits) -1.25 -1.74 -1.95 -2.05 -2.68 -2.81

SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between January 2012 and June 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
- For the 3 hours exposure time, the highest concentration analysed was selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%. For the 24 hours exposure time, the highest concentration analysed was selected based on the solubility of the test item in the culture medium.

Conclusions:

An in vitro micronucleus assay with Hyacinth body was performed according to OECD 487 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It was concluded that Hyacinth body is not clastogenic or aneugenic in human lymphocytes.

Executive summary:

An in vitro micronucleus assay was performed with Hyacinth body, according to OECD 487 guideline and GLP principles. Cultured peripheral human lymphocytes were exposed to different concentrations of Hyacinth body (dissolved in DMSO), in the presence and absence of S9-mix. An in vitro micronucleus assay was performed with Hyacinth body, according to OECD 487 guideline and GLP principles. Cultured peripheral human lymphocytes were exposed to different concentrations of Hyacinth body (dissolved in DMSO), in the presence and absence of S9-mix. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control (3 hours exposure time) or was determined by the solubility (24 hours exposure time). In the first cytogenetic assay, Hyacinth body was tested up to and including cytotoxic concentrations of 400 and 450 μg/mL for a 3 h exposure time with a 27 h fixation time in the absence and presence of S9-mix, respectively. In the second cytogenetic assay, Hyacinth body was tested up to and including the precipitating concentration of 150 μg/mL for a 24 h exposure time with a 24 h fixation time in the absence of S9-mix. Reliable positive and negative controls were included. The substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in any of the two experiments. Therefore, Hyacinth body was determined to be not clastogenic. In the first cytogenetic assay, Hyacinth body was tested up to and including cytotoxic concentrations of 400 and 450 μg/mL for a 3 h exposure time with a 27 h fixation time in the absence and presence of S9-mix, respectively. In the second cytogenetic assay, Hyacinth body was tested up to and including the precipitating concentration of 150 μg/mL for a 24 h exposure time with a 24 h fixation time in the absence of S9-mix. Reliable positive and negative controls were included. The substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in any of the two experiments. Therefore, Hyacinth body was determined to be not clastogenic.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2016 - 11 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015. A new version of the guideline has been adopted by OECD. The study procedures described in this report are also in compliance with this new guideline:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 490: "Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene" (adopted 29 July 2016)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
-Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 125, 250, 500, 1000 and 1943 μg/mL
Without S9-mix, 24 hours treatment: 125, 250, 500, 1000 and 1943 μg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 25, 50, 100, 150, 175, 200, 225, 250, 275 and 300 μg/mL
With S9-mix, 3 hours treatment: 10, 50, 100, 200, 250, 300, 315, 330, 350, 365, 380 and 400 μg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus :
Without S9-mix, 3 hours treatment: 50, 100, 150, 175, 200, 250, 275 and 300 μg/mL
With S9-mix, 3 hours treatment: 10, 50, 100, 200, 300, 330, 350 and 365 μg/mL
Experiment 2:
Without S9-mix, 24 hours treatment: 25, 50, 100, 150, 175, 200, 225, 275, 300, 325 and 350 μg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus:
Without S9-mix, 24 hours treatment: 25, 50, 100, 150, 175, 200 and 225 μg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: test item was dissolved in dimethyl sulfoxide. No correction was made for the purity/composition of the test item.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment: With and without S9-mix: 3 hours
Prolonged treatment period: Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)
NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

- OTHER: The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows: MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10^6
Small and large colony mutation frequencies were calculated in an identical manner.

Evaluation criteria:

Acceptability of the assay:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

Data evaluation:
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 x 10^-6.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The pH and osmolarity at a concentration of 512 μg/mL were 7.61 and 0.449 Osm/kg respectively (compared to 7.68 and 0.455 Osm/kg in the solvent control).
- Precipitation: The test item precipitated at concentrations of 1600 and 1940 μg/mL (=0.01 M) directly after adding to the exposure medium and after 5 minutes.

RANGE-FINDING/SCREENING STUDIES:
The relative suspension growth was 28 and 56% at the test item concentration of 250 μg/mL compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively. No cell survival was observed at test item concentrations of 500 μg/mL and above. The relative suspension growth was 14% at the test item concentration of 250 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentrations of 500 μg/mL and above.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment
Mean 894 724 1694
SD 247 200 793
n 81 74 108
Upper control limit
(95% control limits) 1431 1216 4045
Lower control limit
(95% control limits) 356 231 -657

SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between January 2013 and May 2016.

- Negative (solvent/vehicle) historical control data:
Mutation frequency per 106 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment
Mean 83 75 84
SD 22 24 27
n 161 146 210
Upper control limit
(95% control limits) 128 126 141
Lower control limit
(95% control limits) 37 25 28

SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between January 2013 and May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- First mutagenicity test:
In the absence of S9-mix , the dose levels of 25 and 50 μg/mL showed no cytotoxicity. Therefore, the dose level of 25 μg/mL was not regarded relevant for mutation frequency measurement. The dose level of 225 μg/mL was not used for mutation frequency measurement, since this dose level showed an inconsistent RSG. The dose levels of 200 to 315 μg/mL showed similar cytotoxicity. Therefore, the dose levels of 250 and 315 μg/mL were not regarded relevant for mutation frequency measurement. The dose
levels of 380 and 400 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the absence and presence of S9-mix, the relative total growth of the highest test item concentration was 13% compared to the total growth of the solvent controls.
- Second mutagenicity test:
The dose levels of 275 to 350 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The relative total growth of the highest test item was 16% compared to the total growth of the solvent controls.

Conclusions:

A mouse lymphoma assay with Hyacinth body was conducted according to OECD 490 guideline. Based on the results of this study, Hyacinth body is not mutagenic.

Executive summary:

The mouse lymphoma assay with Hyacinth body was conducted according to OECD 490 guideline and GLP principles. In the first experiment, the test item was tested up to and including concentrations of 300 and 365 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 13% in the absence and presence of S9-mix. In the second experiment, the test item was tested up to the concentration of 225 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was 16%. Positive control chemicals, methyl methane sulfonate and cyclophosphamide, induced appropriate responses. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment (3 h incubation time). This result was confirmed in an independent experiment longer in duration (24 h incubation time). In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment (3 h incubation time).

It is concluded that Hyacinth body is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genetic toxicity is based on the results of an Ames test with Hyacinth body #3 supported by a study on Hyacinth body (comparable to OECD 471 guideline with deviations). Furthermore, an in vitro micronucleus test (OECD TG 487) and in vitro mammalian gene mutations assay (OECD TG 490) are available for Hyacinth body itself. First the genotoxicity information on Hyacinth body #3 is presented, followed by the results of the studies performed on Hyacinth body. Thereafter, the read across justification is presented. The read across justification document is also attached in this endpoint summary.

Genetic mutation in bacteria (read across from Hyacinth body #3 / CAS# 7493-57-4)

The mutagenic activity of Hyacinth body #3 was evaluated in accordance with OECD 471 guideline (Ames test) and according to GLP principles, therefore a Klimisch 1 rating was assigned. The test was performed as a standard plate incorporation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the presence and absence of S9-mix, Hyacinth body #3 was toxic towards the strains TA1535 at 1500 µg/plate and towards the strains TA98, TA100, TA102, and TA1537 at 5000 μg/plate. No precipitation was observed at any of the concentrations. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100, TA102), both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study, it is concluded that Hyacinth body #3 is not mutagenic.

In vitro micronucleus

An in vitro micronucleus assay was performed with Hyacinth body, according to OECD 487 guideline and GLP principles. Cultured peripheral human lymphocytes were exposed to different concentrations of Hyacinth body (dissolved in DMSO), in the presence and absence of S9-mix. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control (3 hours exposure time) or was determined by the solubility (24 hours exposure time). In the first cytogenetic assay, Hyacinth body was tested up to and including cytotoxic concentrations of 400 and 450 μg/mL for a 3 h exposure time with a 27 h fixation time in the absence and presence of S9-mix, respectively. In the second cytogenetic assay, Hyacinth body was tested up to and including the precipitating concentration of 150 μg/mL for a 24 h exposure time with a 24 h fixation time in the absence of S9-mix. Reliable positive and negative controls were included. The substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in any of the two experiments. Therefore, Hyacinth body was determined to be not clastogenic.

In vitro gene mutation

The mouse lymphoma assay with Hyacinth body was conducted according to OECD 490 guideline and GLP principles. In the first experiment, the test item was tested up to and including concentrations of 300 and 365 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 13% in the absence and presence of S9-mix. In the second experiment, the test item was tested up to the concentration of 225 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was 16%. Positive control chemicals, methyl methane sulfonate and cyclophosphamide, induced appropriate responses. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment (3 h incubation time). This result was confirmed in an independent experiment longer in duration (24 h incubation time). In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment (3 h incubation time). It is concluded that Hyacinth body is not mutagenic in the mouse lymphoma L5178Y test system.

Hyacinth body (target; CAS no. 2556-10-7) and its gene mutations in bacteria strain TA102 using read across from Hyacinth body #3 (source; CAS no. 7493-57-4)

 

Introduction and hypothesis for the read across

Hyacinth body has benzyl ring with an ethyl chain to which an acetal is attached and another ethyl chain. An Ames test is available for Hyacinth Body. However in this study, one crucial bacterial strain is missing: E. coli or S. typhimurium TA102. Therefore the analogue approach is selected and the genotoxicity of the missing strains is used from Hyacinth body #3. Therefore additional information is used from read across in accordance with Article 13 of REACH where it is said that lacking information can be used by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, SARs, grouping and read-across.

Hypothesis: Hyacinth body is expected to have the same gene mutation profile in bacteria strain TA102 as its analogue Hyacinth body #3.

Available information: For the target substance an Ames test is available but without the TA102 or E.coli strain. For the source substance Hyacinth body #3, a negative Ames test is available including TA102 and fully according to OECD TG 471, Kl. 1). 

Target and Source chemical(s):

Chemical structures of the target chemical (Hyacinth body) and the source chemical (Hyacinth body #3) are shown in Appendix 1, including physico-chemical properties and toxicological bridging information, thought relevant for genetic toxicity.

Purity / Impurities:

Hyacinth body (target) and Hyacinth body #3 (source) are mono-constituents. The impurities in both substances are all below 2 %.

Analogue justification

According to REACH Annex XI, an analogue approach can be used to replace testing when information from different sources provides sufficient evidence. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

Analogue selection: Hyacinth body #3 was selected as an analogue, being the very close analogue (it has just one carbon more than Hyacinth body) and being a substance from IFF portfolio, for which the adequate data are available.

Structural similarities and differences: Hyacinth body (target) and Hyacinth body #3 (source) have the same backbone and the same functional group- an acetal. The difference is that Hyacinth body has an ethyl group attached to the acetal, while Hyacinth body #3 has a propyl group. 

Toxicokinetics: Based on the similarity in chemical structure and physico-chemical properties as presented in the data matrix, the target and the source chemical are expected to have similar toxicokinetics. When the substances hydrolyse similar metabolites will be formed: both will form 2-phenylethanol and acetaldehyde. Hyacinth body will have ethanol as a metabolite while Hyacinth body#3 will generate propanol.

Toxicodynamics, reactivity: The (genotoxic) reactivity is the same between Hyacinth body (target) and Hyacinth body #3 (source) because the acetal's electrophilicity will be very similar when an ethyl or a propyl group is present.

Remaining uncertainties: There are no remaining uncertainties in view of the reasoning above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix in Table 1 to further support the read across between Hyacinth body and Hyacinth body #3.

Conclusions for genotoxicity in bacterial cells: Ames test and specifically TA102

Hyacinth body is negative for TA102 based on read across from Hyacinth body #3, which does not induce reverse mutations in bacteria and specifically in TA102, both with and without metabolic activation.

 

Data matrix for the read across from Hyacinth body #3 to Hyacinth body

CHEMICAL NAME	Hyacinth body	Hyacinth body #3
	Target	Source
Molecular structure
CAS	2556-10-7	7493-57-4
REACH registration	For 2018	For 2018
Einecs	219-868-9	231-327-9
Tanimoto*	1	0.93
Molecular weight	194.28	208.30
Physico-chemical properties
Appearances	Liquid	Liquid
Water solubility at 20˚C(mg/L)	152.3 (C) 453 (IFF, 2015)	49.14 (C) 86.0 (IFF, 2015)
Log Kow	2.91 (C) 3.5 (IFF, 2015)	3.40 (C) 4.1 (IFF, 2015)
Human health
Ames test	Negative for tested strains: TA1535, TA1537, TA98, TA100	Negative for tested strains: TA1535, TA1537, TA98, TA100, TA102

*The Tanimoto similarity of the selected analogue to Hyacinth body was calculated using Chemmine tools: http://chemmine.ucr.edu/similarity/

C – calculated values from EPISuite

 

Justification for classification or non-classification

Based on the available information in the dossier, Hyacinth body does not need to be classified for genotoxicity according to EU CLP (EC No. 1272/2008 and its amendments).